CN111646993A - Marine anti-glioma active substance iso-wall carboline base C, preparation and application thereof - Google Patents
Marine anti-glioma active substance iso-wall carboline base C, preparation and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method and application of isobaric carboline C as a marine anti-glioma active substance, which is characterized in that a rice fermentation product of blue-gray isobaric actinomycetes derived from marine sediments is used for preparing the isobaric carboline C, the blue-gray isobaric actinomycetes are classified and named as actinotechnologies cyanothecoides cyanaurieus ZZ1866, and the preservation number is CCTCC M2020121. The isobaric carboline C obviously inhibits the proliferation of glioma cells and has application prospect in the aspect of preparing anti-glioma medicaments. The chemical structural formula of the isobaric carboline alkali C is as follows:
Description
Technical Field
The invention belongs to the field of medicines, and relates to a compound isobaric carboline C (actinoallobarboline C) with anti-glioma activity, which is obtained from a metabolite of actinomycetes cyanosus ZZ1866 of a marine source, a preparation method thereof and application thereof in preparing anti-glioma medicines.
Background
Brain glioma is the most common primary intracranial tumor and has the characteristics of high invasiveness, easy recurrence and the like. Currently, surgical resection combined with chemoradiotherapy is the standard treatment mode for glioma, but the prognosis of patients is poor. Despite the tremendous progress in understanding the basic mechanisms underlying glioma development over the last decades, the therapeutic efficacy of gliomas has not changed significantly. Glioma has no complete envelope, operation is difficult to eradicate, and radiotherapy is easy to cause tumor recurrence due to radiation resistance, so that screening of novel anti-glioma drugs has important significance for clinical treatment of glioma.
The secondary metabolites of the marine actinomycetes are important resources found in antitumor drugs or antitumor drug lead compounds, wherein rare actinomycetes with small detection rate except streptomyces in the actinomycetes can also produce a plurality of active compounds, such as gentamicin, micronomicin, rifamycin and the like, and the number of new compounds obtained from the rare actinomycetes is also in an increasing state in recent years, so that the marine actinomycetes is an important source of the drug lead compounds. Actionalloteichusus is a rare actinomycete discovered in 1984, and is a new genus formally established through reclassification research in 2000. Recent reports have shown that Actinomyces heteroclidinium produces a number of different types of natural active products, including compounds with significant anti-tumor activity. Isobaric carboline C (actinoallobarboline C) is an alkaloid compound with a novel structure separated from a metabolite of actinomycete blue gray isobaric fungus ZZ1866 which is a marine source, has a strong inhibition effect on the proliferation of human glioma U87MG and U251 cells, and has an application prospect in the aspect of preparing anti-glioma medicaments.
Disclosure of Invention
The first object of the present invention is to provide a novel compound isobaric carboline C (actinoallobarbolene C) having the chemical structural formula:
the second purpose of the invention is to provide a preparation method of isobaric carboline alkali C, which is realized by the following steps:
(1) isolation culture of Actinoalloteichous cyanaurieus ZZ1866
Dissolving air-dried seabed sediment with sterilized tap water, shaking by a shaking table overnight, diluting the sample suspension to a certain concentration, uniformly dispersing a certain amount of sample diluent into a culture dish containing a solid culture medium, culturing for a certain time at room temperature, respectively transferring different bacterial colonies into another culture dish containing the solid culture medium, and continuously culturing for a certain time at room temperature. Finally, the well-grown ZZ1866 single colony is inoculated to a slant culture medium for culture and then is stored in a refrigerator at 4 ℃ for later use.
The seabed sediment is obtained from sea area near Putuo island of Zhoushan of Zhejiang, and the concentration of the sample diluent is 1 × 10-4～1×10-2g/mL; the rotating speed of the oscillation is 160-180 rpm; the sample volume of the sample diluent is 200 μ L; the culture dish and the solid culture medium for slant culture are both Gao's agar culture medium; the room temperature culture temperature is 20-28 ℃; the culture time is 7-14 days.
(2) Identification of the Strain of Actinoalloteichous cyanaurieus ZZ1866
The strain ZZ1866 obtained by the separation culture in the step (1) is identified by a 16S rDNA sequence analysis method commonly used in laboratories at present, is determined to be actinomycetes with blue gray and is classified and named Actinoallothecycoris ZZ1866, and is preserved by China center for type culture Collection-Wuhan center with the preservation number of CCTCCNO: M2020121.
(3) Preparation of solid fermentation product of Actinoalloteichous cyanaurieus ZZ1866 from Calicaria laevigata
Inoculating the strain of actinomycetes cyanoristiceusZZ 1866 obtained in the step (2) into a large Erlenmeyer flask containing a certain amount of liquid culture medium, and performing shake culture on a culture solution containing ZZ1866 strain at room temperature for a certain time to obtain a strain solution. And finally transferring the strain liquid into a large triangular flask containing a certain amount of solid rice culture medium, and standing and culturing for a certain time at room temperature to obtain a ZZ1866 solid fermentation product containing an anti-glioma active compound isobaric carboline base C.
The ZZ1866 strain is isobaromyces of isobaroline alkali C of generating antitumor active substance; the liquid culture medium is a Gauss liquid culture medium (20 g of soluble starch, 1 g of potassium nitrate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of sodium chloride, 0.01 g of ferrous sulfate and 1L of natural seawater), and the dosage is 250 mL; the solid culture medium is a rice culture medium (40 g of rice and 60 ml of seawater); the volume of the large triangular culture bottle is 500 mL; the room temperature culture temperature is 20-28 ℃; the culture time of the strains is 6-10 days, and the culture time of the rice culture medium is 60 days.
(4) Extraction, separation and purification of isobaric carboline alkali C
And (3) extracting the solid fermentation product of the strain ZZ1866 obtained in the step (3) with ethyl acetate to obtain an ethyl acetate extract. Separating the ethyl acetate total extract by silica gel column chromatography, gradient eluting with mixed solvent of cyclohexane and ethyl acetate and mixed solvent of ethyl acetate and methanol, collecting eluate, detecting each component by High Performance Liquid Chromatography (HPLC), and mixing components containing the same components to obtain six components (A-F). Separating the component D by octadecylsilane chemically bonded silica (ODS) column chromatography to obtain component D1And D2Component D2Separating with gel Sephadex LH-20 column chromatography to obtain three components D2a～D2c. Finally, component D2bSeparating and purifying by High Performance Liquid Chromatography (HPLC) to obtain pure compound isobaric carboline alkali C.
The ratio of the silica gel amount of the silica gel column chromatography to the sample amount of the upper column is 10-15 g:1.0 g; the gradient of the mixed solvent of cyclohexane and ethyl acetate is 10:1, 5:1, 2:1 and 1:1 (volume ratio), and the gradient of the mixed solvent of ethyl acetate and methanol is 5:1 and 1:1 (volume ratio); the ratio of the dosage of ODS (octadecylsilane chemically bonded silica) for column chromatography to the amount of a sample loaded on the column is 20-30 g:1.0 g; the ratio of the ODS dosage of the gel column Sephadex LH-20 column chromatography to the sample amount of the upper column is 20-30 g:1.0 g; the high performance liquid phase separation conditions comprise a novel constant CXTH-3000 high performance liquid chromatograph and Fuji C18CT-30 column (280 × 30mm,10 μm), methanol and water as mobile phase (82/18, volume ratio), detection wavelength 210nm, and flow rate 10.0 mL/min.
(5) Structural identification of isobaric carboline base C
The structure of the isobaric carboline base C is determined according to the data of ultraviolet spectrum, infrared spectrum, one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectrum and high-resolution mass spectrum (HRESIMS).
The third purpose of the invention is to provide the application of isobaric carboline alkali C in preparing anti-glioma drugs. The isobaric carboline C can obviously inhibit the proliferation of glioma U87MG and U251 cells, and has application prospect in the aspect of preparing drugs for treating glioma.
The invention has the advantages that: (1) the marine blue-gray actinomycetes actinoallothecioides ZZ1866 can produce isobaric carboline base C with glioma-resisting alkaloid compound under the culture condition; (2) the isobaric carboline alkali C is a novel alkaloid compound with a novel structure; (3) the isobaric carboline C can obviously inhibit the proliferation of glioma cells U87MG and U251, and can provide a new candidate drug molecule for the treatment of glioma.
Drawings
FIG. 1 is a colony map of Actinoalloteichous cyanogenes ZZ1866, a blue-gray actinomycetes.
FIG. 2 is a high resolution mass spectrum of isobaric carboline base C.
FIGS. 3 to 4 show the hydrogen spectra of isobaric carboline base C.
FIGS. 5 to 6 show the carbon spectra of isobaric carboline base C.
FIGS. 7 to 8 show the HMBC spectra of isobaric carboline base C.
FIG. 9 is a schematic representation relating to HMBC of isobaric carboline base C.
Detailed Description
The invention is described in further detail below with reference to the figures and examples. However, the present invention is not limited to these examples.
Example 1
1. Isolation culture of Actinoalloteichous cyanaurieus ZZ1866
The seabed sediment obtained from the sea area near the islands of Putuo in Zhoushan, Zhejiang province was dried at 28 ℃ for 8 days. Taking out the dried precipitateThe product 1 g was dissolved in 9mL of sterile water, shaken overnight and made up with sterile tap water to a concentration of 1 × 10- 3g/mL of sample solution. And (3) uniformly dispersing 200 mu L of sample liquid into a culture dish containing the Gauss agar solid culture medium, culturing for 7 days at the temperature of 28 ℃, transferring different colonies into another culture dish containing the Gauss agar solid culture medium, and continuously culturing for 7 days at the temperature of 28 ℃. Finally, the well-grown ZZ1866 single colony is inoculated to a Gauss agar solid slant culture medium for culture and then stored in a refrigerator at 4 ℃ for later use.
2. Identification of the Strain of Actinoalloteichous cyanaurieus ZZ1866
The species of the strain ZZ1866 obtained was identified using 16S rDNA sequence analysis.
2.1 Experimental reagents and instruments
PCR reagents: PrimeSTAR Max DNA Polymerase (TaKaRa), primers (Invitrogen), the primer sequence is:
Marker：DL5000
an experimental instrument: centrifuge, electrophoresis apparatus, PCR apparatus, ABI 3730XL sequencer.
2.2 Experimental procedures
Bacterial genomic DNA extraction
Electrophoretic detection
PCR amplification
PCR reaction System
PCR reaction conditions
c. Electrophoretic detection
d. Sequencing, gel cutting, purifying and sequencing
e. And analyzing the result, namely the splicing sequence.
2.3 results of the experiment
2.3 results of the experiment
The sequences after splicing are:
GGTTGGGCCACGGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAA GGCCCGGGAACGTATTCACCGCAGCGTTGCTGATCTGCGATTACTAGCGACTCCGACTTC ACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTAAGAGATTCGCTCCA CCTTGCGATTTCGCAGCCCTCTGTACCGGCCATTGTAGCATGTGTGAAGCCCTGGACATAA GGGGCATGATGACTTGACCTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCCA TGAGTCCCCACCATTACGTGCTGGCAACATGGAACAAGGGTTGCGCTCGTTGCGGGACTT AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGCACACTGACCTT ACGGAAACCCCATCTCTGAGGCGATCCAGTGCATGTCAAACCCAGGTAAGGTTCTTCGCG TTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGT TTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGAG GACGTGGAAATCCCCCACACCTAGCGCCCACCGTTTACGGCGTGGACTACCAGGGTATCT AATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGACCCGCC TTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTCCACCGCTACACCAGGAATTCCAGTC TCCCCTACCGAACTCAAGTCTGCCCGTATCGACTGCACGCCCACAGTTAAGCTGTAGGTT TTCACAGTCGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACG CTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTGCGCC TACCGTCACTTTCGCTTCTTCGGCGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCC TCACGCGGCGTCGCTGCGTCAGGCTTTCGCCCATTGCGCAATATTCCCCACTGCTGCCTCC CGTAGGAGTCTGGGCCGTATCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTA CCCGTCGTCGCCTTGGTAGGCCATTACCCCACCAACAAGCTGATAGGCCGCGGGCCCATC CTGCACCGCCGGAACTTTCCACCCACCCCCATGCGGGGGAAGGTCGTATCCGGTATTAGC CCCGGTTTCCCGAGGTTATCCCAGAGTGCAGGGCAGGTTGCCCACGTGTTACTCACCCGT TCGCCGCTCGTGTACCCCGAAGGGCCTTACCGCTCGACTTGCATGTGTTAAGCACGCCGC CAGCGTTCGTCCTGAGCA(1401bp)。
the 16S rDNA sequence obtained above was compared with the NCBI GenBank database of the American national institute of health, and the results showed that the 16S rDNA sequence of the strain ZZ1866 has 100% similarity with the 16S rDNA sequence of Actinoalloteichous cyanogenicerieus DSM 40103 in the GenBank database (accession No.: HG 917901.1). Therefore, the actinomyces variotis ZZ1866 obtained by the invention is classified and named Actinoalloteichous cyanogriseus ZZ1866 (attached figure 1), and the strain is preserved by China center for type culture Collection with the preservation number of CCTCC NO: M2020121, the preservation date: 2020.5.15, respectively; and (4) storage address: wuhan, Wuhan university.
3. Preparation of fermentation product of Actinoalloteichous cyanaurieus ZZ1866, a blue-gray actinomycetes
A strain of Actinoalloteichousacyanriseus ZZ1866, which is a blue-gray strain of a solid slant culture medium of Gaulter agar, was inoculated into a 500mL Erlenmeyer flask containing 250mL of a liquid Gaulter medium (20 g of soluble starch, 1 g of potassium nitrate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of dipotassium phosphate, 0.5 g of sodium chloride, 0.01 g of ferrous sulfate, and 1L of natural seawater), and the culture broth containing ZZ1866 strain was subjected to rotary shaking (180rpm) at 28 ℃ for 5 days to obtain a strain liquid. Transferring 5mL of strain liquid into 500mL of triangular flask containing rice solid culture medium (40 g of rice and 60 mL of natural seawater), and standing and culturing at 28 deg.C for 60 days to obtain culture containing isobaric carboline C. In total 200 flasks of rice culture of strain ZZ1866 were prepared for this experiment.
4. Extraction, separation and purification of isobaric carboline alkali C
200 bottles of rice culture of strain ZZ1866 obtained in the above experiment were each extracted 3 times with ethyl acetate, the ethyl acetate extracts were combined and concentrated under reduced pressure to give 47.83 g of total extract. Separating the total extract by silica gel (800 g) column chromatography, gradient eluting with cyclohexane and ethyl acetate mixed solvent (10:1, 5:1, 2:1, 1:1, volume ratio, 1000mL each) and ethyl acetate and methanol mixed solvent (5:1, 1:1, volume ratio, 1000mL each), collecting one component per 500mL, detecting each component by High Performance Liquid Chromatography (HPLC), and combining the components containing the same components to obtain six components (A-F). Fraction D (5 g) was separated by column chromatography on octadecylsilane chemically bonded silica (ODS, 150 g) eluted with 1500mL of 70% and 100% methanol, respectively, to give fraction D1And D2. Component D2Separating with gel Sephadex LH-20(100g) column chromatography, eluting with methanol to obtain three components D2a～D2c. Finally, component D2b(20mg) Separation by a semi-preparative high performance liquid chromatograph (instrument: CXTH-3000 is innovated; a chromatographic column: fuji C18CT-30, 280 × 30mm,10 μm, mobile phase methanol/water, 82/18, detection wavelength 210nm, flow rate 10.0mL/min, obtaining compound isobaric carboline alkali C (5.9mg, retention time 35 min).
5. Structural identification of isobaric carboline base C
Isobaric carboline C (actinoallobarboline C) is white amorphous powder; molecular formula C24H21N3O3；[α]D 20–42°(c 0.28,CHCl3) (ii) a Ultraviolet spectrum: UV (MeOH) lambdamax(log) 241(3.82),290(3.71), 352(3.54),369(3.52) nm; infrared spectrum: IR (MeOH) vmax2932,1723,1675,1612,1391,1266, 747cm–1(ii) a High resolution mass spectrum (fig. 2): HRESIMS M/z [ M + Na ]]+422.1476 (calculation C)24H21N3O3Na, 422.1481),[2M+Na]+821.3066 (calculation C)48H42N6NaO6821.3064). By the passage of compounds1H spectrum (shown in figures 3-4),13The C spectrum (figure 5-6) and the HMBC spectrum (figure 7-8) are analyzed to determine the chemical structure of the isobaric carboline base C, and the isobaric carboline base C is a new compound13C and1the signal attribution of H nuclear magnetic resonance is shown in table I, and the HMBC related schematic diagram is shown in figure 9.
6. Anti-glioma activity of isobaric carboline base C
The inhibitory effect of isobarically carboline base C on the proliferation of glioma cells U87MG and U251 cells was determined by sulforhodamine B (SRB) method, and doxorubicin was used as a positive control. Glioma U87MG and U251 cells were cultured in MEM and DMEM media containing 10% FBS at 37 ℃ and 5% carbon dioxide in an incubator, respectively, and the cells cultured for the third generation were used for the experimental study of the present invention. Inoculating cells in a 96-well plate, adding different concentrations of isobaric carboline alkali C after 24h adherence, staining with SRB after 72h drug treatment, measuring the absorbance at 515nm with an enzyme-labeling instrument, detecting the survival rate of tumor cells, and calculating IC of the isobaric carboline alkali C for inhibiting the proliferation of the tumor cells50The value is obtained. The experimental results show that: isobaric carboline base C for remarkably inhibiting glioma cellsProliferation, IC thereof inhibiting proliferation of glioma U87MG and U251 cells50The values were 2.28 and 11.9. mu.M, respectively (Table II).
TABLE 1 NMR data on 13C and 1H of isobaric carboline base C (solvent: deuterated dimethyl sulfoxide DMSO-d6)
| No. | 13C(150MHz) | 1H(600MHz,J in Hz) | No. | 13C(150MHz) | 1H(600MHz,J in Hz) |
| 1 | 140.8,C | – | 13 | 101.5,C | – |
| 2 | 111.1,CH | 7.73,d(8.0) | 14 | 22.4,CH3 | 2.20,s |
| 3 | 127.4,CH | 7.57,t(8.0) | 15 | 116.1,CH | 5.96,d(9.2) |
| 4 | 121.2,CH | 7.32,t(8.0) | 16 | 129.0,CH | 6.65,d(9.2) |
| 5 | 121.8,CH | 8.26,d(8.0) | 17 | 135.1,C | – |
| 6 | 121.5,C | – | 18 | 128.6,CH | 7.34,d(7.5) |
| 7 | 123.2,C | – | 19 | 128.2,CH | 7.28,t(7.5) |
| 8 | 128.7,C | – | 20 | 127.8,CH | 7.22,t(7.5) |
| 9 | 152.5,C | – | 21 | 128.2,CH | 7.28,t(7.5) |
| 10 | 127.1,C | – | 22 | 128.6,CH | 7.34,d(7.5) |
| 11 | 99.8,CH | 7.88,s | 23 | 49.0,CH3 | 2.94,s |
| 12 | 156.5,C | – | 24 | 31.4,CH3 | 4.31,s |
Epi-di, iso-mural carbolinesInhibitory Activity of base C on glioma cell proliferation (IC)50:μM)
The experimental results show that the isobaric carboline alkali C obviously inhibits the proliferation of glioma cells and has good application potential in the aspect of preparing anti-glioma drugs.
Sequence listing
<110> Zhejiang university
<120> preparation and application of ocean anti-glioma active substance iso-wall carboline base C
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial sequence (Unknow)
<400>1
agagtttgat cctggctcag 20
<210>2
<211>19
<212>DNA
<213> Artificial sequence (Unknow)
<400>2
ggttaccttg ttacgactt 19
<210>3
<211>1401
<212>DNA
<213> blue Gray Allium muranus (Actinoalloceieus cyanaurieus ZZ1866)
<400>3
ggttgggcca cgggcttcgg gtgttaccga ctttcatgac gtgacgggcg gtgtgtacaa 60
ggcccgggaa cgtattcacc gcagcgttgc tgatctgcga ttactagcga ctccgacttc 120
acggggtcga gttgcagacc ccgatccgaa ctgagaccgg ctttaagaga ttcgctccac 180
cttgcgattt cgcagccctc tgtaccggcc attgtagcat gtgtgaagcc ctggacataa 240
ggggcatgat gacttgacct catccccacc ttcctccgag ttgaccccgg cagtctccca 300
tgagtcccca ccattacgtg ctggcaacat ggaacaaggg ttgcgctcgt tgcgggactt 360
aacccaacat ctcacgacac gagctgacga cagccatgca ccacctgcac actgacctta 420
cggaaacccc atctctgagg cgatccagtg catgtcaaac ccaggtaagg ttcttcgcgt 480
tgcatcgaat taatccacat gctccgccgc ttgtgcgggc ccccgtcaat tcctttgagt 540
tttagccttg cggccgtact ccccaggcgg ggcgcttaat gcgttagcta cggcacggag 600
gacgtggaaa tcccccacac ctagcgccca ccgtttacgg cgtggactac cagggtatct 660
aatcctgttc gctccccacg ctttcgctcc tcagcgtcag tatcggccca gagacccgcc 720
ttcgccaccg gtgttcctcc tgatatctgc gcattccacc gctacaccag gaattccagt 780
ctcccctacc gaactcaagt ctgcccgtat cgactgcacg cccacagtta agctgtaggt 840
tttcacagtc gacgcgacaa accgcctacg agctctttac gcccaataat tccggacaac 900
gctcgcaccc tacgtattac cgcggctgct ggcacgtagt tagccggtgc ttcttctgcg 960
cctaccgtca ctttcgcttc ttcggcgctg aaagaggttt acaacccgaa ggccgtcatc 1020
cctcacgcgg cgtcgctgcg tcaggctttc gcccattgcg caatattccc cactgctgcc 1080
tcccgtagga gtctgggccg tatctcagtc ccagtgtggc cggtcgccct ctcaggccgg 1140
ctacccgtcg tcgccttggt aggccattac cccaccaaca agctgatagg ccgcgggccc 1200
atcctgcacc gccggaactt tccacccacc cccatgcggg ggaaggtcgt atccggtatt 1260
agccccggtt tcccgaggtt atcccagagt gcagggcagg ttgcccacgt gttactcacc 1320
cgttcgccgc tcgtgtaccc cgaagggcct taccgctcga cttgcatgtg ttaagcacgc 1380
cgccagcgtt cgtcctgagc a 1401
Claims (4)
1. A marine anti-glioma active substance isobaric carboline base C has a chemical structural formula as follows:
2. the method for preparing isobaric carboline base C according to claim 1, characterized in that it is achieved by the following steps:
(1) preparation of blue gray actinomyces variotii fermentation product
Inoculating strains of the blue gray actinomyces anisomerous into a large triangular flask containing a liquid culture medium, carrying out shake culture on a strain culture solution containing the actinomyces anisomerous at room temperature to obtain a strain solution, transferring the strain solution into a large triangular flask containing a rice solid culture medium, and carrying out standing culture at a specific temperature for a certain time to obtain a culture containing an active substance of the carboline alkali C; the actinomyces variotis is classified and named as actinoallothecus cyanoglriseus ZZ1866, and is preserved by China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2020121 and the preservation date of the actinomyces variotis: 2020.5.15, respectively; and (4) storage address: wuhan, Wuhan university;
(2) extraction, separation and purification of isobaric carboline alkali C
Extracting the solid fermentation product of the strain ZZ1866 obtained in the step (1) with ethyl acetate to obtain an ethyl acetate extract, separating the ethyl acetate total extract by silica gel column chromatography, performing gradient elution with a mixed solvent of cyclohexane and ethyl acetate and a mixed solvent of ethyl acetate and methanol, collecting eluent, detecting each component by high performance liquid chromatography, combining the components containing the same components to obtain six components A-F, and separating the component D by octadecylsilane chemically bonded silica gel column chromatography to obtain a component D1And D2Component D2Separating with gel Sephadex LH-20 column chromatography to obtain three components D2a～D2cAnd finally, component D2bSeparating and purifying by high performance liquid chromatography to obtain pure compound isobaric carboline alkali C;
(3) structural identification of isobaric carboline base C
The structure of the isobaric carboline base C is determined according to the ultraviolet spectrum, the infrared spectrum, the one-dimensional and two-dimensional nuclear magnetic resonance spectrum and the high-resolution mass spectrum data.
3. The method for preparing isobaric carboline base C according to claim 2, characterized in that the ratio of the silica gel amount of the silica gel column chromatography in the step (2) to the sample amount of the upper column is 10-15 g:1.0 g; the gradient volume ratio of the mixed solvent of cyclohexane and ethyl acetate is 10:1, 5:1, 2:1 and 1:1, and the gradient volume ratio of the mixed solvent of ethyl acetate and methanol is 5:1 and 1: 1; the ratio of the ODS dosage of the octadecylsilane chemically bonded silica column chromatography to the sample amount of the upper column is 20-30 g:1.0 g; the ratio of the ODS dosage of the gel column Sephadex LH-20 column chromatography to the sample amount of the upper column is 20-30 g:1.0 g; the high-efficiency liquid phase separation conditions are as follows: innovative constant CXTH-3000 high performance liquid chromatograph, Fuji C18The CT-30 chromatographic column is 280 × 30mm,10 μm, methanol and water are used as mobile phase, the detection wavelength is 210nm, and the flow rate is 10.0 mL/min.
4. The use of the isobaric carboline base C according to claim 1 for the preparation of an anti-glioma medicament.
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| CN113024549A (en) * | 2021-03-11 | 2021-06-25 | 浙江大学 | Marine anti-glioma natural active substance-pronaphthidine A as well as preparation and application thereof |
| CN114292266A (en) * | 2021-06-07 | 2022-04-08 | 曲阜师范大学 | Extraction method and application of anti-tumor compound beta-carbazoline |
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| CN113024549B (en) * | 2021-03-11 | 2022-01-11 | 浙江大学 | Marine anti-glioma natural active substance-pronaphthidine A as well as preparation and application thereof |
| CN114292266A (en) * | 2021-06-07 | 2022-04-08 | 曲阜师范大学 | Extraction method and application of anti-tumor compound beta-carbazoline |
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