CN117384228A - Beta-carboline glucuronide, preparation method and application thereof - Google Patents
Beta-carboline glucuronide, preparation method and application thereof Download PDFInfo
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- CN117384228A CN117384228A CN202311285653.4A CN202311285653A CN117384228A CN 117384228 A CN117384228 A CN 117384228A CN 202311285653 A CN202311285653 A CN 202311285653A CN 117384228 A CN117384228 A CN 117384228A
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- AIFRHYZBTHREPW-UHFFFAOYSA-N cis-beta-Carboline Acid Natural products N1=CC=C2C3=CC=CC=C3NC2=C1 AIFRHYZBTHREPW-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 229930182480 glucuronide Natural products 0.000 title claims abstract description 34
- -1 Beta-carboline glucuronide Chemical class 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 102000004889 Interleukin-6 Human genes 0.000 claims abstract description 12
- 108090001005 Interleukin-6 Proteins 0.000 claims abstract description 12
- 229940100601 interleukin-6 Drugs 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 4
- 241001446247 uncultured actinomycete Species 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 29
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 150000008134 glucuronides Chemical class 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 14
- 241000186361 Actinobacteria <class> Species 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 9
- 238000001704 evaporation Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 3
- 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 229940124599 anti-inflammatory drug Drugs 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 238000004809 thin layer chromatography Methods 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 2
- 239000002609 medium Substances 0.000 claims 2
- 241000222385 Phanerochaete Species 0.000 claims 1
- 238000004440 column chromatography Methods 0.000 claims 1
- 238000005238 degreasing Methods 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 238000010829 isocratic elution Methods 0.000 claims 1
- 238000009630 liquid culture Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 238000007790 scraping Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 8
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 4
- 241001366278 Leptotes marina Species 0.000 abstract 1
- 239000002585 base Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 4
- 239000003513 alkali Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000002038 ethyl acetate fraction Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003430 antimalarial agent Substances 0.000 description 1
- 108010030694 avidin-horseradish peroxidase complex Proteins 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
Classifications
-
- 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
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
-
- 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
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/04—Actinomyces
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical fields of chemical biology and medicinal chemistry, and particularly relates to beta-carboline glucuronide, a preparation method and application thereof. And preparing the beta-carboline glucuronide from the marine blue gray actinomycete fermentation product. The beta-carboline glucuronide can obviously reduce the expression of interleukin 6 and has application prospect in the aspect of preparing anti-inflammatory medicaments. The chemical structural formula of the beta-carboline glucuronide is as follows:
Description
Technical Field
The invention relates to the field of chemical synthesis, in particular to beta-carboline glucuronide, a preparation method and application thereof.
Background
The beta-carboline alkaloid compound has a plurality of biological activities such as anti-tumor, anti-malarial, antibacterial, antiviral and the like, has medicine development potential, and is mainly separated from terrestrial animals, plants and marine invertebrates. Actinomycetes are important drug sources, wherein streptomyces terrestrial is the group which has the longest research history and is repeatedly separated and researched most, and marine actinomycetes are emergency drug sources after the excessive development of the actinomycetes, and in recent years, a plurality of beta-carboline alkali compounds with novel structures and remarkable activity are discovered from marine actinomycetes such as heat-resistant marine actinomycetes marinacteosporis thermolerens and actinomycetes bluish gray heterowall actinomycetes acteoshuascyanogriseus. Glycosylation modification is widely involved in various life processes in nature, the biological activity of various compounds is regulated and controlled, the pharmacological characteristics of small molecular drugs are particularly and obviously influenced, such as the solubility is improved, the absorption and transportation of the drugs are improved, and no report of glycosylation modified beta-carboline alkaloids exists at present, so that the discovery and preparation of beta-carboline glycoside have important significance and medicinal prospect.
Disclosure of Invention
The first object of the invention is to provide a new compound beta-carboline base glucuronide, the molecular formula of which is C29H29N3O9, and the chemical structural formula is as follows:
the second object of the invention is to provide a method for preparing beta-carboline base glucuronide, which is realized by the following steps:
(1) Seed culture:
the actinomycetes gracilis isolated from the southern China sea brown flat sponge was streaked on a solid medium (yeast extract, 4g; malt extract, 10g; glucose, 4g; agar, 18g; tap water, 1L, pH: 7.2-7.4) and cultured at 28℃for 7 days.
(2) Fermentation culture:
the bacterial colony spores were scraped to a liquid medium containing 200mL (yeast extract, 4g; soluble starch, 10g; peptone, 2g; sea salt, 33g; deionized water, 1L, pH: 7.0), and subjected to stationary culture at 28℃for 21 days to obtain a marine rare actinomycete fermentation culture.
(3) And (3) extracting and separating:
filtering the fermentation culture with gauze, extracting the filtrate with equal volume of ethyl acetate for 3 times, concentrating the extract under reduced pressure to obtain extract, ultrasonically extracting mycelium part with 1.2 times of volume of dichloromethane/methanol (1:1, V/V) for 5 times, centrifuging to remove mycelium residues, rotary evaporating supernatant to obtain dry organic solvent, extracting the rest water phase with equal volume of ethyl acetate for 3 times, and rotary evaporating to obtain dry extract. The two extracts are combined into a total extract. The total extract was redissolved in 10 volumes of methanol/water (9:1, V/V), defatted 3 times with equal volumes of petroleum ether, the aqueous layer extracted 3 times with equal volumes of dichloromethane, the remaining aqueous layer extracted 3 times with equal volumes of ethyl acetate, and the organic phase was dried by rotary evaporation to obtain the ethyl acetate fraction. Ethyl acetate fraction was dissolved in DSMO injection reverse phase chromatography (ODS, 15 μm,) Gradient eluting with acetonitrile/water with volume fraction of 10% -100% (0.1% formic acid added to water for 240 min), developing by Thin Layer Chromatography (TLC), mixing similar fractions, performing mass spectrometry, tracking, mixing fractions containing target compound with the volume fraction of 25%, reversed-phase semi-preparing liquid phase, eluting with ODS chromatographic column (XBIdge C18,19×150mm,5 μm) with 35% acetonitrile/water at equal temperature and flow rate of 2.0mL/min, detecting wavelength 288nm, and separating and purifying to obtain beta-carboline base glucuronide (retention time 13.5 min).
The third object of the invention is to provide the application of beta-carboline base glucuronide in preparing anti-inflammatory drugs. The compound beta-carboline alkali glucuronide of the invention can obviously reduce the expression of interleukin 6 (IL-6), and has application prospect in the aspect of preparing anti-inflammatory drugs
The invention has the advantages that: (1) Marine-derived blue gray isophthora can produce beta-carboline base glucuronide under the culture conditions; (2) The beta-carboline base glucuronide is a glycosylated alkaloid compound with a novel structure; (3) The beta-carboline base glucuronide can obviously reduce the expression of interleukin 6 (IL-6), and can provide a new candidate medicine molecule for resisting inflammation.
Drawings
FIG. 1 is a hydrogen spectrum of β -carboline base glucuronide.
FIG. 2 is a carbon spectrum of the beta-carboline base glucuronide.
FIG. 3 is a high resolution mass spectrum of the beta-carboline base glucuronide.
FIG. 4 is a schematic illustration of the structure of β -carboline base glucuronide.
FIG. 5 is a graph showing the results of reducing interleukin 6 expression by beta-carboline base glucuronide.
Detailed Description
The technical scheme of the invention is further described below by specific embodiments with reference to the accompanying drawings:
the following examples of the present invention, which are based on the technical solution of the present invention, illustrate detailed embodiments and specific operation procedures, but the present invention is not limited to these examples.
The invention relates to a novel beta-carboline alkali glucuronide separated from actinomyces blumeris. The present invention may also employ other biological methods for preparing the compounds of the present invention or synthetic methods for preparing the compounds of the present invention.
Example 1
Preparation of the Compounds of the invention
(1) Seed culture:
the actinomycetes gracilis isolated from the southern China sea brown flat sponge was streaked on a solid medium (yeast extract, 4g; malt extract, 10g; glucose, 4g; agar, 18g; tap water, 1L, pH: 7.2-7.4) and cultured at 28℃for 7 days.
(2) Fermentation culture:
the bacterial colony spores were scraped to a liquid medium containing 200mL (yeast extract, 4g; soluble starch, 10g; peptone, 2g; sea salt, 33g; deionized water, 1L, pH: 7.0), and subjected to stationary culture at 28℃for 21 days to obtain a marine rare actinomycete fermentation culture.
(3) And (3) extracting and separating:
filtering the fermentation culture with gauze, extracting the filtrate with equal volume of ethyl acetate for 3 times, concentrating the extractive solution under reduced pressure to obtain 12.6g extract, ultrasonically extracting mycelium part with 1.2 times volume of dichloromethane/methanol (1:1, V/V) for 5 times, centrifuging to remove mycelium residues, rotary evaporating supernatant to dry organic solvent, extracting the rest water phase with equal volume of ethyl acetate for 3 times, and rotary evaporating to dry to obtain 4g extract. The two extracts are combined into 16.6g of total extract. The total extract was redissolved in 200mL of methanol/water (9:1, V/V), defatted 3 times with equal volume of petroleum ether, the aqueous layer extracted 3 times with equal volume of dichloromethane, the remaining aqueous layer extracted 3 times with equal volume of ethyl acetate, and the organic phase was dried by rotary evaporation to obtain 420mg of ethyl acetate fraction. Ethyl acetate fraction was dissolved in DSMO injection reverse phase chromatography (ODS, 15 μm,) Gradient elution is carried out by taking acetonitrile/water with gradient fraction of 10% -100% as mobile phase (0.1% formic acid is added into water for 240 min), separating into component Fr.4 (6 mg, volume fraction of 25%) and preparing liquid phase in reverse phase, ODS chromatographic column (XBridge C18,19×150mm,5 μm) is eluted with acetonitrile/water with gradient of 35%, flow rate of 2.0mL/min, detection wavelength of 288nm, separating and purifying to obtain beta-carboline base glucuronide (tR 13.5min,1.8 mg).
Example 2
Identification of the Compounds of the invention
The compound of the invention determines the plane structure and absolute configuration through 1DNMR, 2DNMR, HRESIMS and X-ray single crystal diffraction (CuK alpha), and is a novel compound, and the physicochemical properties and the physicochemical property and nuclear magnetic resonance data of the novel compound are as follows: pale yellow fine wool-like crystals;
(c0.27,MeOH)；mp289℃；UV(MeOH)λmax(logε),223(2.98),268(2.72),288(2.85),373(1.92)nm；IRνmax3482,3328,2921,2852,1650,1681,1650,1509,1458,1420,1361,1292,1183,1155,1016,958,906,828,792,747(FigS3)；HRESIMS,m/z564.1981[M+H]++ (calcdford 29h29n3o9, 564.1982); 1HNMR (DMSO-d 6, 600 MHz) and 13CNMR (DMSO-d 6,150 MHz) data are shown in Table 1.
Table 1 nuclear magnetic resonance spectroscopy data for beta-carboline base glucuronide
Example 3
Anti-inflammatory experiments for preparing Compounds of the invention
The anti-inflammatory activity of LPS-induced mouse macrophage RAW264.7 is studied by adopting an inflammation model, and the cytokine level is detected by an ELISA method. The levels of IL-6 and tumor necrosis factor alpha (TNF-alpha) in cells were detected by a sandwich method according to the instructions of the ELISA kit of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). Prior to ELISA detection, the captured antibodies were diluted as described with 1-fold buffer. 100 μl of capture antibody solution was added to all wells of a 96-well plate. The well plate was sealed and incubated overnight (16-18 h) at 2℃and 8 ℃. All reagents were warmed to room temperature before use and washed 4 times with at least 300 μl of wash buffer per well, and subsequent washes were performed in the same manner. To block non-specific binding and reduce background, 200 μl of 1X assay diluent a was added per well. The plates were sealed and incubated for 1 hour at room temperature on a shaker. The plate was washed 4 times. The sample was diluted with 1X assay dilution a and 100. Mu.L/well was added to the corresponding well. The well plate was sealed, incubated for 2 hours at RT and the plate was washed 4 times. 100 mu L of diluted antibody solution to be detected is added into each hole, a hole sealing plate is subjected to shaking incubation for 1h at RT, and the plate is washed for 4 times. 100 μl of diluted Avidin-HRP solution was added to each well, the plates were closed, incubated for 30min with shaking at room temperature, and the plates were washed 5 times. 100. Mu.L of the freshly prepared TMB substrate solution was added, incubated in the dark for 20 minutes, and 100. Mu.L of stop solution was added to each well and the reaction stopped. The absorbance at 450nm was read within 15 minutes. Experimental results show that the beta-carboline alkali glucuronide has the advantages of obviously reducing the expression of interleukin 6 at the concentration of 20 mu M, and having good application potential in the aspect of preparing anti-inflammatory medicaments.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (7)
1. The beta-carboline glucuronide is characterized by having a molecular formula of C29H29N3O9, and a chemical structural formula of the beta-carboline glucuronide is as follows:
2. a method for preparing beta-carboline glucuronide, which is characterized by comprising the following steps:
step 1: seed culture:
streaking actinomycetes gracilis separated from Phanerochaete brown, and culturing at 28deg.C for 7 days;
step 2: fermentation culture:
scraping colony spores to 200mL of liquid culture medium, and standing and culturing at 28 ℃ for 21 days to obtain a marine rare actinomycete fermentation culture;
step 3: and (3) extracting and separating:
filtering the fermentation culture with gauze, extracting the filtrate with equal volume of ethyl acetate for 3 times, concentrating the extract under reduced pressure to obtain extract, ultrasonically extracting mycelium part with 1.2 times of volume of dichloromethane and methanol for 5 times, centrifuging to remove mycelium residues, rotary evaporating supernatant to obtain dry organic solvent, extracting the rest water phase with equal volume of ethyl acetate for 3 times, rotary evaporating to obtain dry extract; combining the two extracts to obtain a total extract; dissolving the total extract in 10 times of methanol and water, degreasing with equal volume of petroleum ether for 3 times, extracting water layer with equal volume of dichloromethane for 3 times, extracting the rest water layer with equal volume of ethyl acetate for 3 times, and rotary evaporating to obtain dry organic phase to obtain ethyl acetate part; dissolving the ethyl acetate part in DSMO, injecting into a reverse phase pressure column chromatography, performing gradient elution by taking acetonitrile/water with the volume fraction of 10% -100% as a mobile phase, performing color development according to thin layer chromatography, combining similar fractions, performing mass spectrum tracking, combining fractions containing target compounds, wherein the elution volume fraction is 25%, performing reverse phase semi-preparation liquid phase, performing isocratic elution by using an ODS chromatographic column with the volume fraction of 35% acetonitrile/water, the flow rate is 2.0mL/min, detecting the wavelength of 288nm, and performing separation and purification to obtain the beta-carboline base glucuronide.
3. The method for preparing beta-carboline glucuronide according to claim 1, which is characterized in that: the solid medium comprises: yeast extract, 4g; malt extract, 10g; glucose, 4g; agar, 18g; tap water, 1l, ph:7.2-7.4.
4. The method for preparing beta-carboline glucuronide according to claim 1, which is characterized in that: the liquid medium comprises: yeast extract, 4g; soluble starch, 10g; peptone, 2g; sea salt, 33g; deionized water, 1l, ph:7.0.
5. the method for preparing beta-carboline glucuronide according to claim 1, which is characterized in that: the volume ratio of the dichloromethane to the methanol is 1:1.
6. The method for preparing beta-carboline glucuronide according to claim 1, which is characterized in that: the volume ratio of methanol to water was 9:1.
7. Use of a β -carboline glucuronide as claimed in claim 1, wherein: the application of beta-carboline glucuronide in preparing anti-inflammatory drugs reduces the expression of interleukin 6 (IL-6).
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| CN202311285653.4A CN117384228A (en) | 2023-10-07 | 2023-10-07 | Beta-carboline glucuronide, preparation method and application thereof |
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| CN202311285653.4A CN117384228A (en) | 2023-10-07 | 2023-10-07 | Beta-carboline glucuronide, preparation method and application thereof |
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