CN105112322B - Grey mold quinone A and B and preparation method thereof and medical usage - Google Patents
Grey mold quinone A and B and preparation method thereof and medical usage Download PDFInfo
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Abstract
The present invention is grey mold quinone A and B and preparation method thereof and medical usage, provides the streptomyces griseus P82SMLY detached from marine sediment, deposit number CCTCC NO：M 2015386.It is noval chemical compound that the present invention obtains two kinds of marine natural products grey mold quinone A with anticol matter tumor activity and grey mold quinone B, the grey mold quinone A and grey mold quinone B using bacterial strain extraction classification.Grey mold quinone A and grey mold quinone B provided by the invention has the characteristics that significantly inhibit a variety of brain glioblastoma cell proliferation, and can significantly induce brain glioblastoma cell apoptosis, therefore can be applied in terms for the treatment of glioma drug is prepared.The chemical structural formula of grey mold quinone A and grey mold quinone B is：
Description
Technical field
The invention belongs to field of medicaments, are related to biologically active new marine natural products grey mold quinone A
(streptoanthraquinone A) and grey mold quinone B (streptoanthraquinone B) and preparation method thereof, Yi Ji
Prepare the purposes in antitumor drug.
Background technology
Malignant tumour (cancer) endangers seriously the health and lives of the mankind, is the highest disease of world today's death rate,
It is also first cause of the death of urban and rural residents of China.Chemotherapy is the main method of drug therapy after current total surgical resection in the world,
But chemotherapeutic toxicity is larger, and offer limited effectiveness.Although molecular targeted agents opened up a new way for the treatment of cancer, it is clinical
Curative effect simultaneously falls flat, and cancer cell being continuously increased for tumor drug resistance of confrontation also seriously affects existing drug
The effect of.Obviously, it clinically needs to research and develop structure novel, good effect and safe new type antineoplastic medicine.Naturally
Product, particularly marine natural products are the resource treasure-houses of new type antineoplastic medicine research and development.
Invention content
The object of the present invention is to provide actinomycetes strain of the separation with anticol matter tumor activity from marine sediment
P82SMLY, the strain classification are named as streptomyces griseus P82SMLY（Streptomyces griseus P82SMLY）,
By China typical culture collection center preservation, deposit number CCTCC NO：M 2015386, preservation day 2015.6.19 protect
Hide address：Wuhan, China.
The bacterial strain is actinomyces streptomyces griseusStreptomyces griseusP82SMLY passes through following step
Suddenly it is separately cultured and obtains：
(1) streptomyces griseusStreptomyces griseusP82SMLY's is separately cultured
Air dried marine sediment strain cultivation liquid is taken to dilute, a concentration of 0.1-0.001 g/ after dilution
ML takes a certain amount of sample diluting liquid to evenly spread in the culture dish containing solid medium, cultivates one at ambient temperature
After fixing time, different bacterium colonies is transferred to respectively in another culture dish containing solid medium, is continued at ambient temperature
Cultivate certain time.Well-grown single bacterium colony (P82SMLY) is finally inoculated into slant medium culture and is placed on 4 DEG C of ice
Case saves backup.
The marine sediment is obtained from Zhoushan Of Zhejiang Province Area of The East China Sea；The strain cultivation liquid every 100
The composition of mL is：1.5 grams of glucose, 1.5 grams of glycerine, 1.5 grams of malt extracts, 2.5 grams of yeast extracts, 0.5 gram of junket egg
Casamino acid and 0.1 gram of calcium carbonate, or the fluid nutrient medium of other compositions of Suitable strains P82SMLY growths；The sample
The sampling amount of product dilution is 100-300；Solid medium contained by the culture dish is bacteria Agr (Bacto-agar)
Culture medium or other solid mediums；The slant medium synthesizes slant medium or other solid slope cultures for Gao Shi
Base；The incubated at room temperature temperature is 20-30 DEG C；The incubation time is 7-15 days.
(2) streptomyces griseusStreptomyces griseusThe strain idenfication of P82SMLY
Above-mentioned steps (1) are separately cultured the 16S rDNA sequence analysis sides that the bacterial strain of gained is generally used with current laboratory
The type of method identification bacterial strain P82SMLY, is determined as streptomyces griseus, Classification And Nomenclature isStreptomyces griseus
P82SMLY, by China typical culture collection center preservation-Wuhan, China Wuhan University, deposit number CCTCC NO：M
2015386。
Second object of the present invention is to provide two kinds of marine natural products grey mold quinone A with anticol matter tumor activity
(streptoanthraquinone A, 1) and grey mold quinone B (streptoanthraquinone B, 2), the grey mold quinone A
It is noval chemical compound with grey mold quinone B, their chemical structural formula is：
Third object of the present invention is to provide the preparation method of grey mold quinone A and grey mold quinone B, by marine actinomycete grey chain
MouldStreptomyces griseusP82SMLY is generated, and is realized by following steps：
(1) grey mold quinone A and grey mold quinone B producing strains streptomyces griseusesStreptomyces griseusP82SMLY ferments
The preparation of bacterium solution
Take streptomyces griseusStreptomyces griseusThe colony inoculation of P82SMLY is to containing a certain amount of liquid
In the big conical flask of bacterium culture medium, the culture solution containing P82SMLY strains is rotated to shaking culture one at ambient temperature
To prepare strain liquid after fixing time.Strain liquid is finally transferred to the big conical flask containing a certain amount of liquid fermentation medium,
Rotation shaking fermented and cultured after a certain period of time, obtains the P82SMLY zymocyte liquids with antibacterial activity at ambient temperature.
The formula of the liquid spawn culture medium per 1000mL is:20.0 grams of starch, 1.0 grams of potassium nitrate, 0.5 gram of phosphorus
Sour hydrogen dipotassium, 0.5 gram of magnesium sulfate, 0.5 gram of sodium chloride and 0.01 gram of ferric sulfate, amount used 150-300mL；The liquid hair
Ferment culture medium is liquid Gao Shi synthetic medias, and amount used is 250-500 mL;The big triangle culture bottle for 500 or
1000 mL；The incubated at room temperature temperature is 20-30 DEG C；The rotary speed of the rotation shaking is 160-200 rpm；Institute
The incubation time stated is 5-15 days.
(2) extraction separation and purification of grey mold quinone A and grey mold quinone B
The zymocyte liquid of bacterial strain P82SMLY is extracted with ethyl acetate to obtain acetic acid ethyl ester extract, then uses octadecyl
Silane group silica gel (ODS) column chromatography for separation is eluted with 70%, 85% and 100% methanol, obtains three components respectively.Component 3
Continue to be detached with efficient liquid phase (HPLC), obtain grey mold quinone A and grey mold quinone B.
The ODS dosages of the column chromatography and the sample size ratio of upper amount are 40-60 g: 1.0 g；The efficient liquid phase
Separation condition is:1260 high performance liquid chromatographs of Agilent, 1260 DAD detectors of Agilent, Agilent Zorbax
SB-C18Chromatographic column (250 9.4 mm, 5 ), methanol/water (85/15) be mobile phase, 26 DEG C of column temperature, Detection wavelength
238 nm, flow velocity are 1.0 mL/min.
(3) Structural Identification of grey mold quinone A and grey mold quinone B
The structure of grey mold quinone A and grey mold quinone B be ultraviolet spectra according to them, peacekeeping two dimension NMR spectra and high score
It distinguishes mass spectrometric data and identifies.
Fourth object of the present invention is to provide grey mold quinone A and grey mold quinone B answering in treatment glioma drug is prepared
With.The grey mold quinone A and grey mold quinone B can significantly inhibit the proliferation of a variety of brain glioblastoma cells, significantly induce glioma thin
The apoptosis of born of the same parents.
The drug for grey mold quinone A or grey mold quinone B activity ingredient is independent or both grey mold quinone A and grey mold quinone B are shared or
Grey mold quinone A and grey mold quinone B are with other medicines or together with active ingredient, the drug with pharmaceutically acceptable carrier composition.Institute
The dosage form for stating drug is：Liquid preparation, solid pharmaceutical preparation, capsule preparations, sustained release preparation, nanometer formulation.
The present invention is separately cultured from marine sediment to active material producing strains streptomyces griseusStreptomyces griseusP82SMLY, and two new bioactive substance grey mold quinone A are found that from the culture solution of the bacterial strain
(streptoanthraquinone A) and grey mold quinone B (streptoanthraquinone B).Grey mold quinone A and grey mold quinone B are shown
It writes and inhibits a variety of brain glioblastoma cell proliferation, and can significantly induce brain glioblastoma cell apoptosis, so, grey mold quinone A and grey mold quinone B
The application prospect having had in terms for the treatment of glioma drug is prepared.
Description of the drawings
The uv-spectrogram of Fig. 1 grey mold quinones A (Streptoanthraquinone A, 1).
The hydrogen spectrum of Fig. 2 grey mold quinones A (Streptoanthraquinone A, 1).
The carbon spectrum of Fig. 3 grey mold quinones A (Streptoanthraquinone A, 1).
Fig. 4 and Fig. 5 grey mold quinones A's (Streptoanthraquinone A, 1)1H-1H COSY are composed.
The hsqc spectrum of Fig. 6-8. grey mold quinones A (Streptoanthraquinone A, 1).
The HMBC spectrums of Fig. 9-13. grey mold quinones A (Streptoanthraquinone A, 1).
The HRESIMS spectrums of Figure 14 grey mold quinones A (Streptoanthraquinone A, 1).
The uv-spectrogram of Figure 15 grey mold quinones B (Streptoanthraquinone B, 2).
The hydrogen spectrum of Figure 16 grey mold quinones B (Streptoanthraquinone B, 2).
The carbon spectrum of Figure 17 grey mold quinones B (Streptoanthraquinone B, 2).
Figure 18-20. grey mold quinones B's (Streptoanthraquinone B, 2)1H-1HCOSY is composed.
The hsqc spectrum of Figure 21 grey mold quinones B (Streptoanthraquinone B, 2).
The HMBC spectrums of Figure 22-25. grey mold quinones B (Streptoanthraquinone B, 2).
The NOESY spectrums of Figure 26 grey mold quinones B (Streptoanthraquinone B, 2).
The HRESIMS spectrums of Figure 27 grey mold quinones B (Streptoanthraquinone B, 2).
Figure 28 grey mold quinone A (1) and main COSY, HMBC and NOE correlations of grey mold quinone B (2).
Figure 29 grey mold quinones A inhibits the activity of glioma cell.
Figure 30 grey mold quinones B inhibits the activity of glioma cell.
Figure 31 grey mold quinones A and B induce brain glioblastoma cell apoptosis.
Specific embodiment
The present invention is described in further detail below in conjunction with the drawings and specific embodiments, but that the present invention is not restricted to these is real
Apply example.
Grey mold quinone A and grey mold quinone B producing strains streptomyces griseusesStreptomyces griseusThe separation training of P82SMLY
It supports
Air dried 3 grams of marine sediment is taken to be diluted to a concentration of 0.01 g/mL with strain cultivation liquid.Liquid
The composition of every 100 mL of bacterium culture medium is：1.5 grams of glucose, 1.5 grams of glycerine, 1.5 grams of malt extracts, 2.5 grams of yeast
Extract, 0.5 gram of casamino acid and 0.1 gram of calcium carbonate.Take 200Sample diluting liquid evenly spread to containing bacterium fine jade
In the culture dish of fat (Bacto-agar) solid medium, after cultivating 10 days at ambient temperature, different bacterium colonies is turned respectively
It moves on in another culture dish containing bacteria Agr (Bacto-agar) solid medium, continues culture 7 days at ambient temperature.
Well-grown single bacterium colony (P82SMLY) is finally inoculated into Gao Shi synthesis slant mediums, it is standby to be placed in 4 DEG C of refrigerators preservations
With.
Grey mold quinone A and grey mold quinone B producing strains streptomyces griseusesStreptomyces griseusThe strain mirror of P82SMLY
It is fixed
The type of bacterial strain P82SMLY is obtained using the identification of 16S rDNA sequence analysis methods.
2.1 experiment reagents and instrument
PCR reagent：EX Taq enzymes (TaKaRa), dNTP (TaKaRa), primer (Invitrogen synthesis), primer sequence
Row are:TACGGYTACCTTGTTACGACTT and AGAGTTTGATCMTGGCTCAG
Marker：DL5000
Laboratory apparatus：Centrifuge, electrophoresis apparatus, PCR instrument, ABI 3730XL sequenators.
2.2 experimental procedure
Bacterial genomes DNA is extracted
Electrophoresis detection
PCR amplification
A. PCR reaction systems
10×Ex Taq buffer 2.0μl
2.5mM dNTP Mix 1.6μl
5p Primer 1 0.8μl
5p Primer 2 0.8μl
Template 0.5μl
5u Ex Taq 0.2μl
ddH2O 14.1μl
Total volume 20.0μl
B. PCR reaction conditions
C. electrophoresis detection
D. it is sequenced:Cut glue purification sequencing
E. analysis result:Splice sequence.
2.3 experimental result
Spliced sequence is：TGCAGTCGAACGATGAAGCCCTTCGGGGTGGATTAGTGGCG
AACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAAC
ACTCTGTCCCGCATGGGACGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGG
GTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCA
GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATG
ACGGCC-TTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCC-
GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCG
TAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTG
TGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGA
TCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCG
TAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGG
GAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGA
CGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTCGGTAT
ACAGGTGGTGCATGG-CTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTT
CTGTGTTGCCAGCATGCCCTTCGGGG-TGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGG
ACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATGC
CGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGT
TGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAA
AGTCGGTAACACCCGAAGCCGGTGGCCCAACCCC
16S rDNA sequences achieved above compared with the NCBI GenBank databases of U.S. NIH, the result shows that:
In the 16S rDNA sequences of bacterial strain P82SMLY and GenBank librariesStreptomyces griseusStrain CB00830's
16S rDNA sequences have 100% similitude (accession number: KF9817 31.1).Therefore, the marine bacteria strain that the present invention is obtained
P82SMLY Classification And Nomenclatures are streptomyces griseusStreptomyces griseusP82SMLY.Streptomyces griseus
(Streptomyces griseusP82SMLY) by China typical culture collection center preservation（Wuhan, China Wuhan is big
It learns）, deposit number CCTCC NO：M 2015386.
Grey mold quinone A and grey mold quinone B producing strains streptomyces griseusesStreptomyces griseusP82SMLY zymocyte liquids
Preparation
Take streptomyces griseusStreptomyces griseusThe colony inoculation of P82SMLY is to the liquid containing 200 mL
In the big conical flask of bacterium culture medium, the culture solution containing P82SMLY strains is rotated (180 under room temperature at 28 DEG C
Rpm) shaking culture obtains strain liquid after 5 days.Strain liquid is transferred to 500 mL containing 250 mL liquid Gao Shi synthetic medias
Conical flask after rotation (180 rpm) shaking is cultivated 10 days under room temperature at 28 DEG C, obtains having antibacterial activity
P82SMLY zymocyte liquids.
The extraction separation and purification of grey mold quinone A and grey mold quinone B
The zymocyte liquid (30.0 liters) that above-mentioned experiment 3 obtains is extracted with ethyl acetate 3 times, 10. liters every time, merges acetic acid
Ethyl ester extract liquor, through being concentrated under reduced pressure to give acetic acid ethyl ester extract (5.98 grams).The extract is bonded with octadecylsilane
Silica gel (ODS, 300 grams) column chromatography for separation is successively eluted with 70%, 85% and 100% methanol, is collected eluent respectively and is depressurized dense
Contracting obtains 70%, 85% and 100% methanol eluate, three components.Component three (100% methanol eluate, 50 mg) is detached with HPLC
Purify (instrument：Agilent 1260；Chromatographic column：Agilent Zorbax SB-C18, 2509.4 mm, 5；Mobile phase:
Methanol/water：85/15；Column temperature：26℃；Detection wavelength：238 nm；Flow velocity：1.0 m, L/min), obtain grey mold quinone A (1,30.6
mg, t R16.46 min) and grey mold quinone B (2,11.8 mg,t R 21.45 min)。
According to the ultraviolet spectra of grey mold quinone A and grey mold quinone B (attached drawing 1 and 15), optical rotational activity spectrum,1H composes (attached drawing 2 and 16),13C
Spectrum (attached drawing 3 and 17),1H-1H COSY spectrums (attached drawing 4,5,18,19 and 20), hsqc spectrum (attached drawing 6,7,8 and 21), HMBC
Spectrum (attached drawing 9,10,11,12,13,22,23,24 and 25), NOESY spectrums (attached drawing 26) and high resolution mass spectrum (attached drawing 14
With 27), the Structural Identification of grey mold quinone A and grey mold quinone B are noval chemical compound, and main COSY, HMBC and NOE correlation is referring to attached
Figure 28.
The physicochemical property of grey mold quinone A：Burgundy powder；Molecular formula C27H25NO8; []D 25 + 63.7 (c 0.50,
DMSO); UV (MeOH): 240, 327, 443 nm;High resolution mass spectrum (HRESIMS) ism/z [M+Na]+
514.1457 (calculated value C27H25NNaO8, 514.1478)；13C and1H NMR signals assignments are shown in Table one.
The physicochemical property of grey mold quinone B：Burgundy powder；Molecular formula C28H22O8; []D 25 + 51.9 (c 0.50,
DMSO); UV (MeOH): 221, 237, 327, 445 nm;High resolution mass spectrum (HRESIMS) ism/z [M+Na]+
509.1233 (calculated value C28H22NaO8, 509.1212)；13C and1H NMR signals assignments are shown in Table two.
It is noval chemical compound to determine the grey mold quinone A and grey mold quinone B, their chemical structural formula is：
。
The bioactivity research of grey mold quinone A and grey mold quinone B
5.1. grey mold quinone A and grey mold quinone B inhibits the effect of tumor cell proliferation
Rat brain glioma C6 cells and human glioma U251 cells with DMEM and 10% FBS culture mediums at 37 DEG C and
Cultivated in the incubator of 5% carbon dioxide, and human glioma U87-MG and HSG-44 cell respectively with MEM culture mediums and
RPMI-1640 culture mediums are cultivated in the incubator of 37 DEG C and 5% carbon dioxide, and the cell by three generations's culture is for the present invention
Experimental study.
Tumor cell survival is measured with Sulforhodamine B (SRB) method, adriamycin (Doxorubicin) is for positive drug
Control.Cell inoculation adds in the grey mold quinone A of various concentration or grey mold quinone B in 96 orifice plates after adherent 24 h.72 h of drug-treated
It is dyed afterwards with SRB, measures the absorbance value at 515 nm with microplate reader, detect the survival rate of tumour cell, calculate grey mold quinone A
Inhibit the IC of glioma with grey mold quinone B50Value.The experimental results showed that：Grey mold quinone A and grey mold quinone B significantly inhibit colloid
The proliferation of oncocyte, IC50Value is respectively 0.71-3.93With 4.64-10.48(table three, 29 He of attached drawing
Attached drawing 30).
5.2. the effect of grey mold quinone A and grey mold quinone B inducing apoptosis of tumour cell
It is thin to grey mold quinone A and grey mold quinone B induction people's gliomas U251 with the bis- staining analysis methods of Annexin V-FITC/PI
The effect of born of the same parents' apoptosis has carried out quantitative analysis.By glioma U251 cells grey mold quinone A (0.71) or grey mold quinone B
(4.65 ) processing 36 hours after, collect 1106A cell.Cell is dispersed in again after being washed with cold PBS buffer solution
100 Contain 5Annexin V-FITC and 1In the combination buffer of 100 μ g/ml PI working solutions.Cell exists
400 are added in after hatching 15 minutes at room temperatureCombination buffer, with its fluorescence (excitation wavelength of flow cytomery：488
nm；Launch wavelength：530 nm and 575 nm).The experimental results showed that：Compared with control group U251 Apoptosis (3.58%), ash
Mould quinone A and grey mold quinone B processing after 36 h, can cause respectively Apoptosis of Human Glioma U 251 cell rate increase 38.76 % and
36.67 % (referring to attached drawing 31), the lower left corner is normal cell in figure, and the lower right corner is viable apoptotic cell, and the upper right corner is withered for late period
Cell is died, the upper left corner is non-viable non-apoptotic cell.
The above result shows that：Grey mold quinone A and grey mold quinone B significantly inhibits brain glioblastoma cell proliferation, and significantly induces brain glue
Matter apoptosis of tumor.It is answered so the related drugs prepared by grey mold quinone A and grey mold quinone B have in terms of the treatment of glioma
Use prospect.
<110>Zhejiang University
<120>Grey mold quinone A and B and preparation method thereof and medical usage
<160> 3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
TACGGYTACCTTGTTACGACTT
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
AGAGTTTGATCMTGGCTCAG
<210> 3
<211> 1255
<212> DNA
<213>Streptomyces griseus (Streptomyces griseus P82SMLY)
<220>
<222> (1)…(1255)
<440>1
ACTGCGTCAC CTTCGAAGCT CCCTCCCACA AGGGGTTGGG CCACCGGCTT 50
CGGGTGTTAC CGACTTTCGT GACGTGACGG GCGGTGTGTA CAAGGCCCGG 100
GAACGTATTC ACCGCAGCAA TGCTGATCTG CGATTACTAG CAACTCCGAC 150
TTCATGGGGT CGAGTTGCAG ACCCCAATCC GAACTGAGAC CGGCTTTTTG 200
AGATTCGCTC CGCCTCGCGG CATCGCAGCT CATTGTACCG GCCATTGTAG 250
CACGTGTGCA GCCCAAGACA TAAGGGGCAT GATGACTTGA CGTCGTCCCC 300
ACCTTCCTCC GAGTTGACCC CGGCAGTCTC CTGTGAGTCC CCATCACCCC 350
GAAGGGCATG CTGGCAACAC AGAACAAGGG TTGCGCTCGT TGCGGGACTT 400
AACCCAACAT CTCACGACAC GAGCTGACGA CAGCCATGCA CCACCTGTAT 450
ACCGACCACA AGGGGGGCAC CATCTCTGAT GCTTTCCGGT ATATGTCAAG 500
CCTTGGTAAG GTTCTTCGCG TTGCGTCGAA TTAAGCCACA TGCTCCGCTG 550
CTTGTGCGGG CCCCCGTCAA TTCCTTTGAG TTTTAGCCTT GCGGCCGTAC 600
TCCCCAGGCG GGGAACTTAA TGCGTTAGCT GCGGCACCGA CGACGTGGAA 650
TGTCGCCAAC ACCTAGTTCC CAACGTTTAC GGCGTGGACT ACCAGGGTAT 700
CTAATCCTGT TCGCTCCCCA CGCTTTCGCT CCTCAGCGTC AGTAATGGCC 750
CAGAGATCCG CCTTCGCCAC CGGTGTTCCT CCTGATATCT GCGCATTTCA 800
CCGCTACACC AGGAATTCCG ATCTCCCCTA CCACACTCTA GCTAGCCCGT 850
ATCGAATGCA GACCCGGGGT TAAGCCCCGG GCTTTCACAT CCGACGTGAC 900
AAGCCGCCTA CGAGCTCTTT ACGCCCAATA ATTCCGGACA ACGCTTGCGC 950
CCTACGTATT ACCGCGGCTG CTGGCACGTA GTTAGCCCGG CGCTTCTTCT 1000
GCAGGTACCG TCACTTTCGC TTCTTCCCTG CTGAAAGAGG TTTACAACCC 1050
GAAAGGCCGT CATCCCTCAC GCGGCGTCGC TGCATCAGGC TTTCGCCCAT 1100
TGTGCATATT CCCCACTGCT GCCTCCGTAG AGTCTGGTCG TGTCTTCAGT 1150
TCCAGTGTGT GTCGCCCCTC CCTCAGCGCC TACCGTCGGT CGCCTTGTTA 1200
GCATACCCCA CACCAGCTGA ATGCGCGGCT CATCCTTCCA CCCGCCGGAG 1250
CATCA 1255
Claims (5)
1. a kind of streptomyces griseus bacterial strain P82SMLY, it is characterised in that:The streptomyces griseus bacterial strain is preserved in Chinese Typical Representative training
Object collection is supported, deposit number is CCTCC NO：M 2015386, Classification And Nomenclature are：Streptomyces griseus P82SMLY
（Streptomyces griseus P82SMLY）, preservation day：On June 19th, 2015.
2. a kind of streptomyces griseus bacterial strain P82SMLY according to claim 1, which is characterized in that pass through following steps point
It is obtained from culture：
(1) streptomyces griseusStreptomyces griseusP82SMLY's is separately cultured
Air dried marine sediment strain cultivation liquid is taken to dilute, a concentration of 0.1-0.001 g/mL after dilution,
Sample diluting liquid is taken to evenly spread in the culture dish containing solid medium, at ambient temperature incubation time after 7-15 days,
Different bacterium colonies is transferred to respectively in another culture dish containing solid medium, continues to cultivate 7-15 at ambient temperature
My god, well-grown single colony inoculation to slant medium culture is finally placed on 4 DEG C of refrigerators and is saved backup；
The marine sediment is obtained from Zhoushan Of Zhejiang Province Area of The East China Sea；Every 100 mL's of the strain cultivation liquid
It forms and is：1.5 grams of glucose, 1.5 grams of glycerine, 1.5 grams of malt extracts, 2.5 grams of yeast extracts, 0.5 gram of casein ammonia
Base acid and 0.1 gram of calcium carbonate, or the fluid nutrient medium of other compositions of Suitable strains P82SMLY growths；The sample is dilute
The sampling amount for releasing liquid is 100-300 mL；Solid medium contained by the culture dish is bacteria Agr Bacto-agar culture mediums
Or other solid mediums；The slant medium synthesizes slant medium or other solid slope culture mediums for Gao Shi；Room
Warm cultivation temperature is 20-30 DEG C；
（2）Strain idenfication：Step (1) is separately cultured to the 16S rDNA sequences that are generally used with current laboratory of bacterial strain of gained
Analysis method is identified, is determined as streptomyces griseus, Classification And Nomenclature isStreptomyces griseusP82SMLY, preservation
In China typical culture collection center, deposit number CCTCC NO：M 2015386.
3. anticol matter tumor activity substance grey mold quinone B (streptoanthraquinone B), which is characterized in that the grey mold
Quinone B has following chemical structural formula：
。
4. the preparation method of anticol matter tumor activity substance grey mold quinone B according to claim 3, which is characterized in that by with
Lower step is realized：
(1) grey mold quinone B producing strains streptomyces griseusesStreptomyces griseusThe preparation of P82SMLY zymocyte liquids
Take streptomyces griseusStreptomyces griseusThe colony inoculation of P82SMLY is to containing liquid spawn culture medium
In big conical flask, the culture solution containing P82SMLY strains is rotated at ambient temperature after shaking is cultivated 5-15 days to prepare
Strain liquid is finally transferred to the big conical flask containing liquid fermentation medium by strain liquid, at ambient temperature rotation shaking hair
After ferment culture 5-15 days, the P82SMLY zymocyte liquids with antibacterial activity are obtained；
The formula of the liquid spawn culture medium per 1000mL is:20.0 grams of starch, 1.0 grams of potassium nitrate, 0.5 gram of phosphoric acid hydrogen
Dipotassium, 0.5 gram of magnesium sulfate, 0.5 gram of sodium chloride and 0.01 gram of ferric sulfate, amount used 150-300mL；The liquid fermentation training
It is liquid Gao Shi synthetic medias to support base, and amount used is 250-500 mL；The big triangle culture bottle is 500 or 1000
mL；Incubated at room temperature temperature is 20-30 DEG C；The rotary speed of the rotation shaking is 160-200 rpm；
The streptomyces griseus bacterial strain is preserved in China typical culture collection center, and deposit number is CCTCC NO：M
2015386, Classification And Nomenclature is：Streptomyces griseus P82SMLY（Streptomyces griseus P82SMLY）, preservation day：
On June 19th, 2015；
(2) extraction separation and purification of grey mold quinone B
The zymocyte liquid of bacterial strain P82SMLY is extracted with ethyl acetate to obtain acetic acid ethyl ester extract, then uses octadecylsilane
Bonded silica gel column chromatography for separation is eluted respectively with 70%, 85% and 100% methanol, obtains three components, and component 3 continues with efficient
Liquid phase separation obtains grey mold quinone B；
The ODS dosages of the column chromatography and the sample size ratio of upper amount are 40-60 g: 1.0 g；The efficient liquid phase separation
Condition is:1260 high performance liquid chromatographs of Agilent, 1260 DAD detectors of Agilent, Agilent Zorbax SB-
C18Chromatographic column：250 × 9.4 mm, 5 μm, methanol/water ratio is for 85/15 as mobile phase, 26 DEG C of column temperature, Detection wavelength
238 nm, flow velocity are 1.0 mL/min；
(3) Structural Identification of grey mold quinone B
The structure of grey mold quinone B is identified by ultraviolet spectra, the NMR spectra of peacekeeping two dimension and high resolution mass spectrum data.
5. grey mold quinone B according to claim 3 is preparing the application in treating glioma drug.
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