CN111747955B - Marine anti-glioma active substance isobaric carboline alkali A, preparation and application thereof - Google Patents
Marine anti-glioma active substance isobaric carboline alkali A, preparation and application thereof Download PDFInfo
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Abstract
The invention provides a marine anti-glioma active substance isobaric carboline alkali A, a preparation method and an application thereof, the isobaric carboline alkali A and the blue gray isobaric actinomycetes are prepared by utilizing rice fermentation products of blue gray isobaric actinomycetes derived from marine sediments, are classified and named as actinotechnologies cyanothrieus ZZ1866, and have a preservation number of CCTCC M2020121. The isobaric carboline alkali A 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 A is as follows:
Description
Technical Field
The invention belongs to the field of medicines, relates to a marine anti-glioma active substance isobaric carboline base A, and preparation and application thereof, and relates to a compound isobaric carboline base A (actinoallocatecarboline A) with anti-glioma activity, which is obtained from a metabolite of actinomyces anollothecus cyanogeneus ZZ1866, a preparation method thereof, and application thereof in preparation of anti-glioma drugs.
Background
Glioma is one of the most common primary central nervous system tumors occurring in the glial tissue of the brain, and the incidence rate of glioma is on an increasing trend year by year. At present, the standard treatment mode for patients with glioma is surgical resection, temozolomide and radiotherapy are given, then temozolomide is given for auxiliary treatment, but glioma grows invasively, and no obvious limit exists between tumor tissues and normal tissues, so focal tissues cannot be completely removed by surgical resection, radiotherapy and chemotherapy means can only act on a certain proportion of tumor cells, most of chemical anti-cancer drugs are blocked outside by a blood brain barrier and a blood vessel brain tumor barrier, the drug concentration at the part where glioma occurs is too low to kill the tumor cells, and the increase of the administration concentration brings serious side effects to the normal tissues. Although molecularly targeted drugs open a new path for tumor treatment, glioma is also extremely susceptible to relapse after treatment due to the presence of tumor stem cells and drug resistance problems. Clearly, there is a clinical need for new anti-glioma drugs with better efficacy, higher safety and unique mechanism of action.
The marine organisms are not only rich in various resources, but also evolve a unique metabolic pathway to resist the extreme environment of the ocean, so that the marine natural products have structural specificity different from that of the land. The marine microorganisms gradually become the research center of gravity in marine organism research due to the characteristics of special habitat, genetic controllability, amplifiable fermentation and the like, wherein actinomycetes are an important source for finding antitumor drugs or drug lead compounds.
Disclosure of Invention
The first purpose of the invention is to provide a novel compound isobaric carboline A (actinoallobaroline A), which has the chemical structural formula:
the second purpose of the invention is to provide a preparation method of isobaric carboline alkali A, 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 the sea area near the Putuo island in Zhoushan, Zhejiang; the concentration of the sample diluent is 1 x 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 actinomyces varioti, is classified and named as Actinoallothecus cyanogriseus ZZ1866, and is preserved by China center for type culture Collection-Wuhan center with the preservation number of CCTCC NO: M2020121.
(3) Preparation of solid fermentation product of Actinoalloteichous cyanaurieus ZZ1866 from Calicaria laevigata
Inoculating the strain of actinomycetes cyanotrichus cyanriseus ZZ1866 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 the isobaric carboline alkali A compound with glioma resistance activity.
The ZZ1866 strain is isobaromyces of isobaroline alkali A 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 A
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 component C with octadecylsilane chemically bonded silica (ODS) column chromatography, respectively using 70% and 1%Elution with 00% methanol gave fraction C1And C2. Finally, component C2Separating and purifying by High Performance Liquid Chromatography (HPLC) to obtain pure compound isobaric carboline alkali A.
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 high performance liquid phase separation conditions comprise a novel constant CXTH-3000 high performance liquid chromatograph and Fuji C18CT-30 column (280X 30mm,10 μm), methanol and water as mobile phase (90/10, volume ratio), detection wavelength 210nm, flow rate 10.0 mL/min.
(5) Structural identification of isobaric carboline base A
The structure of the isobaric carboline alkali A is determined by combining methods such as ultraviolet spectrum, infrared spectrum, one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectrum, high resolution mass spectrum (HRESIMS) data, single crystal X-ray diffraction and the like.
The third purpose of the invention is to provide the application of isobaric carboline alkali A in preparing anti-glioma drugs. The isobaric carboline A can obviously inhibit the proliferation of glioma U87MG and U251 cells, and has application prospect in the aspect of preparing drugs for treating glioma.
Isobaric carboline A (actinoallococcus verticillatus ZZ1866) is an alkaloid compound with a novel structure separated from a metabolite of actinoallococcus cyaneus ZZ1866 of a marine source, has a remarkable inhibition effect on the proliferation of U87MG and U251 glioma cells, and has an application prospect in the aspect of preparing anti-glioma medicaments.
The invention has the advantages that: (1) the marine blue-gray actinomycetes actinoallothecium cyanogenicum ZZ1866 can produce isobaric carboline alkali A with glioma-resisting alkaloid compound under the culture condition; (2) the isobaric carboline alkali A is a novel alkaloid compound with a novel structure; (3) the isobaric carboline A 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 A.
FIGS. 3 to 4 show the hydrogen spectra of isobaric carboline base A.
FIGS. 5 to 6 show the carbon spectra of isobaric carboline base A.
FIG. 7 is a COSY spectrum of isobaric carboline base A.
FIGS. 8 to 9 show the HMQC spectra of isobaric carboline base A.
FIGS. 10 to 11 show the HMBC spectra of isobaric carboline base A.
FIG. 12 is a structural diagram of single crystal X-ray diffraction of isobaric carboline base A.
FIG. 13 is a diagram showing the correlation of COSY and HMBC of isobaric carboline base A.
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. Dissolving 1 g of dried sediment in 9mL of sterile water, shaking overnight, and making into 1 × 10 concentration with sterilized tap water- 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
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)。
a comparison of the NCBI GenBank database of the national institute of health, USA, shows that the 16S rDNA sequence of strain ZZ1866 has 100% similarity with the 16S rDNA sequence of Actinoalloteichous cyanogenes DSM 40103 in the GenBank database (accession: 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 Actinoalloteichous cyanaurieus 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 the 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 A as anti-glioma active substance. In total 200 flasks of rice culture of strain ZZ1866 were prepared for this experiment.
4. Extraction, separation and purification of isobaric carboline alkali A
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 C (3 g) was separated by column chromatography on octadecylsilane chemically bonded silica (ODS, 90 g) eluted with 1500mL of 70% and 100% methanol, respectively, to give fraction C1And C2. Finally, component C2(30mg) was separated by semi-preparative HPLC (Instrument: Innovative constant CXTH-3000; column: Fuji C)18CT-30,280 is multiplied by 30mm,10 mu m; mobile phase: methanol/water, 90/10; the detection wavelength is 210nm, the flow rate is 10.0mL/min), and the compound isobaric carboline alkali A (10.8mg, retention time 32min) is obtained.
5. Structural identification of isobaric carboline base A
Isobaric carboline A (actinoallobarboline A) is colorless needle crystal; molecular formula C24H21N3O3(ii) a The melting point is 291-294 ℃; [ alpha ] to]D 20–90°(c 0.12,CHCl3) (ii) a Ultraviolet spectrum: UV (MeOH) lambdamax(log ε)235(3.79), 294(3.92),363(3.74) nm; infrared spectrum: IR (MeOH) vmax 2926,1713,1664,1610,1372,1327, 1262,735,692cm–1(ii) a High resolution mass spectrum (fig. 2): HRESIMS M/z [ M + Na ]]+422.1479 (calculation C)24H21N3O3Na,422.1481),[2M+Na]+821.3068 (calculation C)48H42N6NaO6821.3064). By the passage of compounds1H spectrum (shown in figures 3-4),13C spectrum (figure 5-6), COSY spectrum (figure 7), HMQC spectrum (figure 8-9), HMBC spectrum (figure 10-11) and X-ray single crystal diffraction (figure 12) analysis determine the chemical structure of the isobaric carboline alkali A, and the isobaric carboline alkali A is a new compound13C and1the signal attribution of H nuclear magnetic resonance is shown in table I, and the relative schematic diagram of COSY and HMBC is shown in figure 13.
Epi-isobaric carboline base A13C and1h NMR data (solvent: deuterated dimethyl sulfoxide DMSO-d)6)
6. Anti-glioma activity of isobaric carboline base A
The inhibitory effect of isobarically carboline alkali A on the proliferation of glioma cells U87MG and U251 cells was determined by a 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 A after adherence for 24h, staining with SRB after drug treatment for 72h, measuring absorbance at 515nm with an enzyme-labeling instrument, detecting survival rate of tumor cells, and calculating IC of isobaric carboline alkali A for inhibiting tumor cell proliferation50The value is obtained. Fruit of Chinese wolfberryThe test result shows that: isobaric carboline A significantly inhibits glioma cell proliferation, and IC thereof inhibits glioma cell proliferation of U87MG and U25150The values were 4.47 and 17.4. mu.M, respectively (Table II).
Epidiasporine inhibition of glioma cell proliferation (IC)50:μM)
The experimental results show that the isobaric carboline alkali A 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> ocean anti-glioma active substance isobaric carboline alkali A, preparation and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 20
<212> DNA
<213> Artificial sequence (Unknow)
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<213> Artificial sequence (Unknow)
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ggttaccttg ttacgactt 19
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<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 (3)
1. A preparation method of a marine anti-glioma active substance isobaric carboline alkali A is characterized by comprising the following steps:
(1) preparation of blue gray actinomyces variotii fermentation product
Inoculating strains of blue gray actinomyces isoborneus into a large triangular flask containing a liquid culture medium, carrying out shake culture on a strain culture solution containing actinomyces isoborneus 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 isobaric carboline alkali A; calycoma pallidum classified and namedActinoalloteichus cyanogriseusZZ1866, has already been preserved by China center for type culture Collection, the preserving number is CCTCC M2020121, the preserving date: 2020.5.15, respectively; and (4) storage address: wuhan, Wuhan university;
(2) extraction, separation and purification of isobaric carboline alkali A
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 total ethyl acetate 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, separating the component C by octadecylsilane chemically bonded silica gel column chromatography, and eluting with 70% and 100% methanol respectively to obtain a component C1And C2And finally, component C2Separating and purifying by high performance liquid chromatography to obtain pure compound isobaric carboline alkali A;
(3) structural identification of isobaric carboline base A
The structure of the isobaric carboline alkali A is determined according to the ultraviolet spectrum, the infrared spectrum, the one-dimensional and two-dimensional nuclear magnetic resonance spectrum, the high-resolution mass spectrum data and the single crystal X-ray diffraction method;
the chemical structural formula of the isobaric carboline alkali A is as follows:
2. the method for preparing the isobaric carboline base A according to claim 1, wherein 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:1.0g, the gradient volume ratio of the mixed solvent of cyclohexane and ethyl acetate is 10:1, 5:1, 2:1, 1:1, and the gradient volume ratio of the mixed solvent of ethyl acetate and methanol is 5:1, 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 high-efficiency liquid phase separation conditions are as follows: innovative constant CXTH-3000 high performance liquid chromatograph, Fuji C18CT-30 chromatographic column 280
30mm,10 μm, methanol and water as mobile phase, detection wavelength 210nm, flow rate 10.0 mL/min.
3. The use of isobaric carboline base a prepared by the method of claim 1 in the preparation of anti-glioma medicaments.
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