CN113444131B - N-acetylglucosamine compounds, and preparation method and application thereof - Google Patents
N-acetylglucosamine compounds, and preparation method and application thereof Download PDFInfo
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- CN113444131B CN113444131B CN202110716707.2A CN202110716707A CN113444131B CN 113444131 B CN113444131 B CN 113444131B CN 202110716707 A CN202110716707 A CN 202110716707A CN 113444131 B CN113444131 B CN 113444131B
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- acetylglucosamine
- dichloromethane
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- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical class CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 26
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims abstract description 19
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims abstract description 19
- 229950006780 n-acetylglucosamine Drugs 0.000 claims abstract description 19
- -1 N-acetylglucosamine compound Chemical class 0.000 claims abstract description 17
- 201000010099 disease Diseases 0.000 claims abstract description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 241000203233 Aspergillus versicolor Species 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 11
- 230000004151 fermentation Effects 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 241000607471 Edwardsiella tarda Species 0.000 claims abstract description 8
- 241000607618 Vibrio harveyi Species 0.000 claims abstract description 8
- 241000191938 Micrococcus luteus Species 0.000 claims abstract description 7
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims abstract description 7
- 241000233866 Fungi Species 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 10
- 239000003960 organic solvent Substances 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000003208 petroleum Substances 0.000 claims description 8
- 238000010898 silica gel chromatography Methods 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 7
- 235000002639 sodium chloride Nutrition 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 238000004809 thin layer chromatography Methods 0.000 claims description 7
- 239000000287 crude extract Substances 0.000 claims description 6
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- FHHZOYXKOICLGH-UHFFFAOYSA-N dichloromethane;ethanol Chemical compound CCO.ClCCl FHHZOYXKOICLGH-UHFFFAOYSA-N 0.000 claims description 4
- 239000000499 gel Substances 0.000 claims description 4
- 230000002441 reversible effect Effects 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- UAJGYNKUMWOQFQ-UHFFFAOYSA-N dichloromethane;propan-1-ol Chemical compound ClCCl.CCCO UAJGYNKUMWOQFQ-UHFFFAOYSA-N 0.000 claims description 2
- BLEBFDYUDVZRFG-UHFFFAOYSA-N dichloromethane;propan-2-ol Chemical compound ClCCl.CC(C)O BLEBFDYUDVZRFG-UHFFFAOYSA-N 0.000 claims description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 150000001875 compounds Chemical class 0.000 abstract description 15
- 230000002401 inhibitory effect Effects 0.000 abstract description 8
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 3
- 239000000022 bacteriostatic agent Substances 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 241000228212 Aspergillus Species 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
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- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical group [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 4
- 238000009360 aquaculture Methods 0.000 description 4
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- 238000002474 experimental method Methods 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
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- 238000012360 testing method Methods 0.000 description 3
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- 230000008569 process Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010048723 Multiple-drug resistance Diseases 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- WBKFWQBXFREOFH-UHFFFAOYSA-N dichloromethane;ethyl acetate Chemical compound ClCCl.CCOC(C)=O WBKFWQBXFREOFH-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- PSLIMVZEAPALCD-UHFFFAOYSA-N ethanol;ethoxyethane Chemical compound CCO.CCOCC PSLIMVZEAPALCD-UHFFFAOYSA-N 0.000 description 1
- YKWNUSJLICDQEO-UHFFFAOYSA-N ethoxyethane;propan-2-ol Chemical compound CC(C)O.CCOCC YKWNUSJLICDQEO-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
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- 229930000044 secondary metabolite Natural products 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
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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
- C07H1/06—Separation; Purification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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Abstract
The invention relates to an N-acetylglucosamine compound and a preparation method and application thereof, belonging to the technical field of bacteriostatic agents and microbial medicines, wherein the preparation method comprises the following steps: fermenting and culturing marine fungus Aspergillus versicolor (Aspergillus) M-7-SW9 in PDB fungus culture medium to obtain fermentation product, and extracting and separating the fermentation product to obtain new N-acetylglucosamine compounds. According to the N-acetylglucosamine compound and the preparation method and application thereof, the minimum inhibitory concentration of the compound on aquatic disease bacteria, namely Edwardsiella tarda and Vibrio harveyi is 1.0 microgram/ml, and the compound also has antibacterial activity on aquatic disease bacteria, namely micrococcus luteus and Vibrio parahaemolyticus, and can be used for preparing a medicament for resisting aquatic disease bacteria.
Description
Technical Field
The invention relates to a novel compound and a preparation method and application thereof, in particular to an N-acetylglucosamine compound extracted and separated from a fermentation product of Aspergillus versicolor (Aspergillus versicolor) M-7-SW9, a preparation method and application thereof, belonging to the technical field of bacteriostatic agents and microbial medicines.
Background
With the expansion of aquaculture scale and the use of a large amount of antibiotics in the aquaculture process, the multiple drug resistance phenomenon of bacteria which are diseases of aquaculture animals is increasingly common, and the use of a large amount of chemical synthetic drugs brings serious pollution to water and soil and causes harm to human and biological health, so that a new idea is provided for solving the problem of bacterial disease control in the current aquaculture process by developing marine organism-derived natural drugs.
The marine fungi can produce secondary metabolites with abundant chemical structure diversity and remarkable biological activity, and are important sources of medicinal lead compounds; n-acetylglucosamine is a basic composition unit of a plurality of important polysaccharides in biological cells, has a plurality of important physiological functions in organisms, also has the functions of diminishing inflammation, resisting tumors and resisting oxidation, is clinically a medicament for treating osteoarthritis and rheumatoid arthritis, and has wide application in the fields of foods, medicines, cosmetics and the like.
According to literature research, the N-acetylglucosamine compounds related to the invention are novel compounds and are only reported before.
Disclosure of Invention
The purpose of the invention is: in order to overcome the defects in the prior art, an N-acetylglucosamine compound and a preparation method and application thereof are provided.
The technical scheme for solving the technical problems is as follows:
an N-acetylglucosamine compound is shown as formula (I), and has molecular formula C 9 H 15 NO 5 ；
A preparation method of N-acetylglucosamine based compounds comprises the following steps:
1) Fermenting and culturing marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9 in a PDB fungus culture medium, repeatedly soaking and extracting a fermentation product by using an organic solvent extracting solution, extracting by using ethyl acetate, combining extracting solutions, and concentrating to obtain a fermentation crude extract; the Aspergillus versicolor (Aspergillus versicolor) M-7-SW9 is preserved in the China Center for Type Culture Collection (CCTCC) at 25 months and 4 months in 2021, and the preservation number is CCTCC NO: m2021454;
2) Subjecting the crude extract obtained in the step 1) to silica gel column chromatography, performing gradient elution by using an organic solvent elution system, collecting eluent, and detecting the eluent by thin layer chromatography;
3) Collecting the eluted components in the step 2), sequentially carrying out reverse phase silica gel column chromatography, gel column chromatography and thin layer chromatography separation and purification, and collecting the components with Rf value of 0.6-0.7 separated and purified by thin layer chromatography to obtain the N-acetylglucosamine compound shown in the formula (I); the volume ratio of the thin-layer chromatography separation and purification developing agent is 2:1 petroleum ether-ethyl acetate.
Furthermore, the organic solvent extracting solution in the step 1) is one or more of dichloromethane, ethyl acetate, methanol, ethanol, propanol or isopropanol.
Further, the organic solvent elution system in the step 2) is one or more of petroleum ether-ethyl acetate, petroleum ether-ethanol, petroleum ether-propanol, petroleum ether-isopropanol, dichloromethane-ethyl acetate, dichloromethane-methanol, dichloromethane-ethanol, dichloromethane-propanol or dichloromethane-isopropanol; the volume ratio of the organic solvent elution system is 50-0:1.
further, the volume ratio of the reverse phase silica gel column chromatography eluent in the step 3) is 4-0:1 water-methanol or water-ethanol; the volume ratio of the gel column chromatography eluent is 2-0:1 dichloromethane-methanol or dichloromethane-ethanol.
Furthermore, the PDB fungus culture medium formula is as follows: each liter of liquid culture medium contains 200 g of potato powder, 20 g of glucose, 5g of peptone, 3 g of yeast extract and 35 g of sea salt.
Furthermore, the N-acetylglucosamine compound shown in the formula (I) can be used for preparing a novel medicament for resisting aquatic disease bacteria; the bacteria are Edwardsiella tarda, vibrio harveyi, micrococcus luteus or Vibrio parahaemolyticus of aquatic disease bacteria.
The beneficial effects of the invention are: the method comprises the steps of culturing and fermenting Aspergillus versicolor M-7-SW9 in a culture medium, extracting and separating the obtained fermentation product to obtain a new N-acetylglucosamine compound, wherein no report of the compound on the inhibition activity of aquatic disease bacteria is found at present, no related medicine is found on the market, and the minimum inhibitory concentration of the compound on aquatic disease bacteria, namely Edwardsiella tarda and Vibrio harveyi is 1.0 microgram/ml, and the compound has the inhibitory activity on aquatic disease bacteria, namely Micrococcus luteus and Vibrio parahaemolyticus, and can be used for preparing the medicine for resisting aquatic disease bacteria.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The invention obtains the compound N-acetylglucosamine compound as shown in the following examples by culturing and fermenting Aspergillus versicolor M-7-SW9 in a culture medium, extracting and separating the fermentation product, wherein the chemical structure is shown as formula (I):
a marine water sample collected in a Bohai sea area is separated from Aspergillus versicolor (Aspergillus versicolor) M-7-SW9, and is characterized by comprising the following steps: white aerial hyphae grow on the PDA culture medium, gray green spores grow on the PDA culture medium, white aerial hyphae grow on the PDB liquid culture medium in the early stage, and the mycoderm turns dark green in the later stage.
Example 1
A preparation method of N-acetylglucosamine compounds as shown in a formula (I):
(1) Inoculating marine fungus Aspergillus versicolor (Aspergillus versicolor) M-7-SW9 (size is 1.5 cm multiplied by 1.5 cm) growing in a plate culture medium into 250mL of PDB liquid culture medium, fermenting for 7 days at 28 ℃ by using a shaker (200 revolutions per minute), inoculating the obtained bacterial liquid into 300L of PDB liquid culture medium, fermenting for 7 days by using a 500L fermentation tank, soaking and extracting the fermentation product by using ethanol, repeatedly extracting by using ethyl acetate, combining the extraction liquid and concentrating to obtain a fermentation crude extract;
the PDB liquid culture medium comprises the following formula: 200 g of potato powder, 20 g of glucose, 5g of peptone, 3 g of yeast extract and 35 g of sea salt in each liter of water.
(2) The crude extract was subjected to reduced pressure silica gel column chromatography and purified with a gradient of 50:1 to 1:1 (v/v) petroleum ether-ethyl acetate and a gradient of 50:1 to 1:1 (v/v) dichloromethane-methanol is used as a solvent to carry out gradient elution in sequence, and dichloromethane-methanol 10 is collected: 1 (v/v), performing reverse phase silica gel column chromatography, and separating the eluate from the column by chromatography at a ratio of 4-0:1 water-methanol elution; collecting the water-methanol 1:1 (v/v), and further purified by High Performance Liquid Chromatography (HPLC) using water-methanol 1:1 (v/v) is a mobile phase, an absorption peak of 18.5min at the wavelength of 210nm is collected to obtain a purified target compound, and the structure of the purified target compound is identified as shown in a formula (I):
the compound has the following physicochemical and spectral characteristics:
a white powdery solid; specific rotation angle [ alpha ]] 20 D -18 (c 0.15, meoh); hydrogen nuclear magnetic resonance spectrum (solvent is deuterated methanol) delta H 5.69 (dd, 4.1, 0.7), 6.33 (br s), 4.87 (ddd, 3.5,1.5, 0.7), 3.55 (dd, 5.2, 3.5), 3.66 (dd, 10.7, 3.5), 3.58 (dd, 10.7, 5.2), 2.05(s), 3.33(s); nuclear magnetic resonance carbon spectrum (solvent is deuterated methanol) delta C 107.5(CH)，133.9(C)，111.7(CH)，86.9(CH)，75.8(CH)，64.4(CH 2 )，172.0(C)，23.1(CH 3 )，53.4(CH 3 ) (ii) a High resolution mass spectrum m/z 240.08519[ M ] +Na ]] + ，C 9 H 15 NO 5 Na + Calculated value 240.084244.
Example 2
Aquatic disease bacteria inhibitor activity:
detecting the activity of the compound shown in the formula (I) against aquatic disease bacteria by using a minimum inhibitory concentration method, and selecting the following 4 aquatic pathogen strains: the antibacterial activity of Edwardsiella tarda, vibrio harveyi, micrococcus luteus and Vibrio parahaemolyticus was tested.
(1) Antibacterial activity test (MIC method):
the Minimum Inhibitory Concentration (MIC), i.e. the lowest concentration of drug that is able to inhibit bacterial growth in vitro. In a 96 micropore plate, medicines with different concentrations are added into a bacterial suspension of bacteria to be detected, observation is carried out after culture, if an indicator bacterium grows in a certain hole, the medicine concentration in the hole cannot inhibit the growth of the bacteria, liquid in the hole is turbid, and the light transmittance is obviously reduced; on the contrary, the liquid in the hole is clear, and the transmittance is not reduced obviously. The lowest sample concentration within the well that completely inhibited the growth of the indicator bacteria was the MIC of the compound.
(2) Preparation of the bacterial suspension
The above test bacteria were inoculated on a medium (TSB medium for Edwardsiella tarda, LB medium for Vibrio harveyi, micrococcus luteus and Vibrio parahaemolyticus, respectively) and cultured at 28 ℃ for 24 hours, and then 4mL of a sterile 0.85% NaCl solution (8.5 g of sodium chloride to 1000mL of water) was aspirated to wash the culture, and the bacteria were gently scraped off with a glass scraper; pipetting an appropriate amount of the bacterial suspension into a sterile test tube with a pipette, then adjusting the bacterial suspension to 0.5 malt turbidity (equivalent to 1.5X 108 CFU/mL) with a 0.85% NaCl solution, and further diluting to 5X 105CFU/mL with a 0.85% NaCl solution;
the 0.5 mcz turbidity scale was: 0.5mL of 0.048mol/L BaCl 2 (1.175％w/v BaCl 2 ·2H 2 O) to 99.5mL of 0.18mol/L (0.36N) H 2 SO 4 (1%) v/v) while stirring continuously to maintain the suspension state.
(3) Preparation of samples
About 1mg of a sample to be tested (the N-acetylglucosamine compound obtained above) and a positive control (chloramphenicol) are respectively dissolved in about 100 μ L of DMSO, and after the mixture is sufficiently and uniformly mixed, the final concentration is 2560 μ g/mL, 50 μ L of sample solution is sucked into another centrifuge tube, and then 50 μ L of DMSO is added, so that a sample solution with the concentration reduced by half is obtained. According to this method, a total of 11 sets of sample solutions (2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5. Mu.g/mL) with successively halved concentrations were obtained.
(4) Blank control: pure solvent (DMSO) to dissolve the sample to be tested was chosen as a blank.
(5) MIC determination procedure
5.1 Adopting sterile operation, respectively adding sample solutions with different concentrations after dilution by multiple times into a sterile 96-well plate, wherein the sample solution is added into the 1 st to 11 th wells, each well is 5 mu L, and the 12 th well is not added with a sample and is used as a growth control.
5.2 95. Mu.L of an indicator suspension corresponding to 0.5 McLeod turbidity was diluted 1000-fold in a liquid medium (TSB medium for Edwardsiella tarda, vibrio harveyi, micrococcus luteus and Vibrio parahaemolyticus LB medium), and then added to a 96-well plate in order to give final concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125. Mu.g/mL in the 1 st to 11 th wells in order. After gently shaking and mixing, the 96-well plate is sealed and placed in an incubator at 28 ℃ for bacterial culture for 24h.
5.3 The absorbance of each well was measured using a microplate reader at a wavelength of 600nm, and the lowest sample concentration at which the growth of the indicator bacteria was completely inhibited in the well was taken as the MIC of the compound. ( Note that: the experiment is meaningful when the obvious growth of the indicator bacteria in the negative control hole is detected; when a single jump hole appears in the experiment, the highest concentration of the drug for inhibiting the growth of the strain is recorded; if a plurality of jump holes appear, the result should not be reported, and the experiment needs to be repeated. )
The experimental result shows that the N-acetylglucosamine compound has stronger inhibitory activity on Edwardsiella tarda and Vibrio harveyi respectively, and MIC values are both 1.0 mug/mL.
The experiment results prove that the compound has a strong inhibiting effect on aquatic disease bacteria, and can be used for preparing a novel aquatic disease bacteria resistant medicament.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (4)
1. A preparation method of N-acetylglucosamine compounds is characterized by comprising the following steps:
1) Fermenting and culturing Aspergillus versicolor M-7-SW9 in PDB fungus culture medium, repeatedly soaking and extracting the fermentation product with organic solvent extract, extracting with ethyl acetate, mixing the extracts, and concentrating to obtain fermented crude extract; the Aspergillus versicolor M-7-SW9 is preserved in China Center for Type Culture Collection (CCTCC) at 25 months and 4 months in 2021, and the preservation number is CCTCC NO: m2021454; the organic solvent extracting solution is one of methanol, ethanol, propanol or isopropanol;
2) Subjecting the crude extract obtained in the step 1) to silica gel column chromatography, performing gradient elution by using an organic solvent elution system, collecting eluent, and detecting the eluent by thin layer chromatography; the organic solvent elution system is one of petroleum ether-ethyl acetate, dichloromethane-methanol, dichloromethane-ethanol, dichloromethane-propanol or dichloromethane-isopropanol; the volume ratio of the organic solvent elution system is 50-0:1;
3) Collecting the eluted components in step 2), sequentially separating and purifying by reverse phase silica gel column chromatography, gel column chromatography and thin layer chromatography, collecting the components with Rf value of 0.6-0.7, and obtaining the N-acetylglucosamine compound shown in formula (I), wherein the molecular formula is C 9 H 15 NO 5 ；
The volume ratio of the thin-layer chromatography separation and purification developing agent is 2:1 petroleum ether-ethyl acetate.
2. The method for producing an N-acetylglucosamine compound according to claim 1, wherein: the volume ratio of the reversed phase silica gel column chromatography eluent in the step 3) is 4-0:1 water-methanol or water-ethanol; the volume ratio of the gel column chromatography eluent is 2-0:1 dichloromethane-methanol or dichloromethane-ethanol.
3. The method for producing an N-acetylglucosamine compound according to claim 1, wherein: the PDB fungus culture medium comprises the following components in percentage by weight: each liter of liquid culture medium contains 200 g of potato powder, 20 g of glucose, 5g of peptone, 3 g of yeast extract and 35 g of sea salt.
4. Use of the N-acetylglucosamine compound prepared by the method for preparing an N-acetylglucosamine compound according to claim 1, wherein: the N-acetylglucosamine compound shown in the formula (I) can be used for preparing medicines for resisting aquatic disease bacteria; the bacteria are Edwardsiella tarda, vibrio harveyi, micrococcus luteus or Vibrio parahaemolyticus of aquatic disease bacteria.
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