CN105112322A - Grisic quinone A, grisic quinone B, and preparation method and medical application of grisic quinone A and grisic quinone B - Google Patents
Grisic quinone A, grisic quinone B, and preparation method and medical application of grisic quinone A and grisic quinone B Download PDFInfo
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Abstract
The invention provides grisic quinone A, grisic quinone B, and a preparation method and medical application of the grisic quinone A and the grisic quinone B. Streptomyces griseus P82SMLY separated from marine sediment is provided, and the preservation number is CCTCC NO: M 2015386. Two marine natural products with antiglioma activity, such as the grisic quinone A and the grisic quinone B, are obtained by extracting and classifying a bacterial strain, and the grisic quinone A and the grisic quinone B are new compounds. The grisic quinone A and the grisic quinone B have a feature of obviously inhibiting multiplication of various kinds of glioma cells, can induce apoptosis of the glioma cells obviously, and accordingly can be used for preparing medicines for treating glioma. The chemical structural formula of the grisic quinone A and the grisic quinone B is as shown in the specification.
Description
Technical field
The invention belongs to field of medicaments, relate to and there is bioactive new marine natural product grey mold quinone A (streptoanthraquinoneA) and grey mold quinone B (streptoanthraquinoneB) and preparation method thereof, and preparing the purposes in antitumor drug.
Background technology
The health and lives harm of malignant tumour (cancer) to the mankind is serious, is first cause of the death of the disease that world today's mortality ratio is the highest, urban and rural residents of Ye Shi China.Chemotherapy is the main method of pharmacological agent after current total surgical resection in the world, but chemotherapeutic toxicity is comparatively large, and offer limited effectiveness.Although molecular targeted agents is the treatment of cancer open new way, its clinical efficacy also falls flat, and the continuous increase of cancer cells antagonism tumor drug resistance also has a strong impact on the curative effect of existing medicine.Obviously, need clinically to research and develop the high new type antineoplastic medicine of novel structure, good effect and security.Natural product, particularly marine natural product are the resource treasure-houses of new type antineoplastic medicine research and development.
Summary of the invention
The object of this invention is to provide the actinomycetes strain P82SMLY being separated from oceanic sediment and there is anticol matter tumor activity, described strain classification called after streptomyces griseus P82SMLY(
streptomycesgriseusp82SMLY), by China typical culture collection center preservation, deposit number CCTCCNO:M2015386, preservation day 2015.6.19, preservation address: Wuhan, China.
Described bacterial strain is actinomycetes streptomyces griseus
streptomycesgriseusp82SMLY, is obtained by following steps separation and Culture:
(1) streptomyces griseus
streptomycesgriseusthe separation and Culture of P82SMLY
Get the dilution of air dried oceanic sediment strain cultivation liquid, concentration after dilution is 0.1-0.001g/mL, get in the culture dish that a certain amount of sample diluting liquid evenly spreads to containing solid medium, after cultivating certain hour at ambient temperature, different bacterium colonies is transferred to another respectively to be contained in the culture dish of solid medium, continues at ambient temperature to cultivate certain hour.Finally well-grown single bacterium colony (P82SMLY) is inoculated into slant medium and cultivates that to be placed on 4 DEG C of Refrigerator stores for subsequent use.
Described oceanic sediment obtains from Zhoushan Of Zhejiang Province Area of The East China Sea; The described every 100mL of strain cultivation liquid consists of: 1.5 grams of glucose, 1.5 grams of glycerol, 1.5 grams of malt extracts, 2.5 grams of yeast extracts, 0.5 gram of casamino acids and 0.1 gram of calcium carbonate, or other liquid nutrient medium formed that Suitable strains P82SMLY grows; The sampling amount of described sample diluting liquid is 100-300
; Solid medium contained by described culture dish is bacteria Agr (Bacto-agar) substratum or other solid medium; Described slant medium is that Gao Shi synthesizes slant medium or other solid slant culture base; Described incubated at room temperature temperature is 20-30 DEG C; Described incubation time is 7-15 days.
(2) streptomyces griseus
streptomycesgriseusthe strain identification of P82SMLY
The bacterial strain of above-mentioned steps (1) separation and Culture gained identifies the kind of bacterial strain P82SMLY with the 16SrDNA sequence analysis method that current laboratory generally uses, be defined as streptomyces griseus, Classification And Nomenclature is
streptomycesgriseusp82SMLY, by China typical culture collection center preservation-Wuhan, China Wuhan University, deposit number CCTCCNO:M2015386.
Second object of the present invention be to provide two kinds of marine natural product grey mold quinone A with anticol matter tumor activity (streptoanthraquinoneA,
1) and grey mold quinone B (streptoanthraquinoneB,
2), described grey mold quinone A and grey mold quinone B is new compound, and their chemical structural formula is:
3rd object of the present invention is to provide the preparation method of grey mold quinone A and grey mold quinone B, by marine actinomycete streptomyces griseus
streptomycesgriseusp82SMLY produces, and is realized by following steps:
(1) grey mold quinone A and grey mold quinone B producing strains streptomyces griseus
streptomycesgriseusthe preparation of P82SMLY zymocyte liquid
Get streptomyces griseus
streptomycesgriseusnutrient solution containing P82SMLY bacterial classification to containing in the large Erlenmeyer flask of a certain amount of liquid spawn culture medium, is rotated jolting and cultivates after certain hour to prepare strain liquid by the colony inoculation of P82SMLY at ambient temperature.Finally strain liquid is proceeded to the large Erlenmeyer flask containing a certain amount of liquid fermentation medium, after rotating jolting fermentation culture certain hour at ambient temperature, obtain the P82SMLY zymocyte liquid with anti-microbial activity.
The formula of the every 1000mL of described liquid spawn culture medium is: 20.0 grams of starch, 1.0 grams of saltpetre, 0.5 gram of dipotassium hydrogen phosphate, 0.5 gram of magnesium sulfate, 0.5 gram of sodium-chlor and 0.01 gram of ferric sulfate, and amount used is 150-300mL; Described liquid fermentation medium is liquid Gao Shi synthetic medium, and amount used is 250-500mL; Described large triangle culturing bottle is 500 or 1000mL; Described incubated at room temperature temperature is 20-30 DEG C; The speed of rotation of described rotation jolting is 160-200rpm; Described incubation time 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 and obtains acetic acid ethyl ester extract, then uses octadecylsilane chemically bonded silica (ODS) column chromatography for separation, uses the methanol-eluted fractions of 70%, 85% and 100% respectively, obtains three components.Component 3 continues to be separated with high performance liquid phase (HPLC), obtains grey mold quinone A and grey mold quinone B.
The ODS consumption of described column chromatography and the sample size ratio of upper amount are 40-60g:1.0g; Described high performance liquid phase separation condition is: Agilent1260 high performance liquid chromatograph, Agilent1260DAD detector, AgilentZorbaxSB-C
18chromatographic column (250
9.4mm, 5
), methanol/water (85/15) is moving phase, column temperature 26 DEG C, determined wavelength 238nm, and flow velocity is 1.0mL/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 is UV spectrum according to them, the NMR spectrum of a peacekeeping two dimension and high resolution mass spectrum data and identify.
4th object of the present invention is to provide the application of grey mold quinone A and grey mold quinone B in preparation treatment cerebral glioma medicine.Described grey mold quinone A and grey mold quinone B significantly can suppress the propagation of multiple brain glioblastoma cell, significantly the apoptosis of induction brain glioblastoma cell.
Described medicine is that grey mold quinone A or grey mold quinone B activeconstituents are independent, or grey mold quinone A and grey mold quinone B share, or grey mold quinone A with grey mold quinone B with other medicines or together with effective constituent, the medicine formed with pharmaceutically acceptable carrier.The dosage form of described medicine is: liquid preparation, solid preparation, capsule preparations, sustained release preparation, nanometer formulation.
The present invention from oceanic sediment separation and Culture to active substance producing strains streptomyces griseus
streptomycesgriseusp82SMLY, and from the nutrient solution of this bacterial strain, found two new biologically active substance grey mold quinones A (streptoanthraquinoneA) and grey mold quinone B (streptoanthraquinoneB).Grey mold quinone A and grey mold quinone B significantly suppresses multiple brain glioblastoma cell to be bred, and can significantly induce brain glioblastoma cell apoptosis, so, the application prospect that grey mold quinone A and grey mold quinone B has had in preparation treatment cerebral glioma medicine.
Accompanying drawing explanation
Fig. 1. the uv-spectrogram of grey mold quinone A (StreptoanthraquinoneA, 1).
Fig. 2. the hydrogen spectrum of grey mold quinone A (StreptoanthraquinoneA, 1).
Fig. 3. the carbon spectrum of grey mold quinone A (StreptoanthraquinoneA, 1).
Fig. 4 and Fig. 5. grey mold quinone A's (StreptoanthraquinoneA, 1)
1h-
1hCOSY composes.
The hsqc spectrum of Fig. 6-8. grey mold quinone A (StreptoanthraquinoneA, 1).
The HMBC spectrum of Fig. 9-13. grey mold quinone A (StreptoanthraquinoneA, 1).
Figure 14. the HRESIMS spectrum of grey mold quinone A (StreptoanthraquinoneA, 1).
Figure 15. the uv-spectrogram of grey mold quinone B (StreptoanthraquinoneB, 2).
Figure 16. the hydrogen spectrum of grey mold quinone B (StreptoanthraquinoneB, 2).
Figure 17. the carbon spectrum of grey mold quinone B (StreptoanthraquinoneB, 2).
Figure 18-20. grey mold quinone B (StreptoanthraquinoneB, 2)
1h-
1hCOSY composes.
Figure 21. the hsqc spectrum of grey mold quinone B (StreptoanthraquinoneB, 2).
The HMBC spectrum of Figure 22-25. grey mold quinone B (StreptoanthraquinoneB, 2).
Figure 26. the NOESY spectrum of grey mold quinone B (StreptoanthraquinoneB, 2).
Figure 27. the HRESIMS spectrum of grey mold quinone B (StreptoanthraquinoneB, 2).
Figure 28. COSY, HMBC and NOE dependency that grey mold quinone A (1) and grey mold quinone B (2) is main.
Figure 29. grey mold quinone A suppresses the activity of glioma cell.
Figure 30. grey mold quinone B suppresses the activity of glioma cell.
Figure 31. grey mold quinone A and B induces brain glioblastoma cell apoptosis.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but the invention is not restricted to these embodiments.
grey mold quinone A and grey mold quinone B producing strains streptomyces griseus
streptomycesgriseusthe separation and Culture of P82SMLY
Getting air dried oceanic sediment 3 grams strain cultivation liquid, to be diluted to concentration be 0.01g/mL.The every 100mL of liquid spawn culture medium consists of: 1.5 grams of glucose, 1.5 grams of glycerol, 1.5 grams of malt extracts, 2.5 grams of yeast extracts, 0.5 gram of casamino acids and 0.1 gram of calcium carbonate.Get 200
sample diluting liquid evenly spread in the culture dish containing bacteria Agr (Bacto-agar) solid medium, cultivate at ambient temperature after 10 days, different bacterium colonies being transferred to respectively another contains in the culture dish of bacteria Agr (Bacto-agar) solid medium, continues cultivation 7 days at ambient temperature.Finally well-grown single bacterium colony (P82SMLY) is inoculated into Gao Shi and synthesizes slant medium, be placed in 4 DEG C of Refrigerator stores for subsequent use.
grey mold quinone A and grey mold quinone B producing strains streptomyces griseus
streptomycesgriseusthe strain identification of P82SMLY
Use the qualification of 16SrDNA sequence analysis method obtain the kind of bacterial strain P82SMLY.
2.1 experiment reagents and instrument
PCR reagent: EXTaq enzyme (TaKaRa), dNTP (TaKaRa), primer (Invitrogen synthesis), primer sequence is: TACGGYTACCTTGTTACGACTT and AGAGTTTGATCMTGGCTCAG
Marker：DL5000
Laboratory apparatus: whizzer, electrophoresis apparatus, PCR instrument, ABI3730XL sequenator.
2.2 experimental procedure
Bacterial genomes DNA extracting
Electrophoresis detection
Pcr amplification
A.PCR reaction system
10×ExTaqbuffer2.0μl
2.5mMdNTPMix1.6μl
5pPrimer10.8μl
5pPrimer20.8μl
Template0.5μl
5uExTaq0.2μl
ddH
2
O14.1μl
Totalvolume20.0μl
B.PCR reaction conditions
C. electrophoresis detection
D. check order: cut glue purification order-checking
E. analytical results: splicing sequence.
2.3 experimental result
Spliced sequence is: TGCAGTCGAACGATGAAGCCCTTCGGGGTGGATTAGTGGCG
AACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAACACTCTGTCCCGCATGGGACGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCC-TTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCC-GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGG-CTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGG-TGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATGCCGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCC
The 16SrDNA sequence more than obtained compares with the NCBIGenBank database of America NI H, and its result shows: in the 16SrDNA sequence of bacterial strain P82SMLY and GenBank storehouse
streptomycesgriseusthe 16SrDNA sequence of strainCB00830 has the similarity (accession number: KF981731.1) of 100%.Therefore, the marine bacteria strain P82SMLY Classification And Nomenclature that the present invention obtains is streptomyces griseus
streptomycesgriseusp82SMLY.Streptomyces griseus (S
treptomycesgriseusp82SMLY) by China typical culture collection center preservation (Wuhan, China Wuhan University), deposit number CCTCCNO:M2015386.
grey mold quinone A and grey mold quinone B producing strains streptomyces griseus
streptomycesgriseusthe preparation of P82SMLY zymocyte liquid
Get streptomyces griseus
streptomycesgriseusnutrient solution containing P82SMLY bacterial classification, in the large Erlenmeyer flask of the liquid spawn culture medium containing 200mL, rotates after (180rpm) jolting cultivates 5 days and obtains strain liquid by the colony inoculation of P82SMLY under 28 DEG C of room temperature conditions.Strain liquid is proceeded to the 500mL Erlenmeyer flask containing 250mL liquid Gao Shi synthetic medium, after rotation (180rpm) jolting cultivates 10 days under 28 DEG C of room temperature conditions, obtain the P82SMLY zymocyte liquid with anti-microbial activity.
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, each 10. liters, combined ethyl acetate extraction liquid, obtains acetic acid ethyl ester extract (5.98 grams) through concentrating under reduced pressure.This extract, with by octadecylsilane chemically bonded silica (ODS, 300 grams) column chromatography for separation, successively uses 70%, 85% and 100% methanol-eluted fractions, collects elutriant respectively and concentrating under reduced pressure obtains 70%, 85% and 100% methanol-eluted fractions thing, three components.Component three (100% methanol-eluted fractions thing, 50mg) HPLC separation and purification (instrument: Agilent1260; Chromatography column: AgilentZorbaxSB-C
18, 250
9.4mm, 5
; Moving phase: methanol/water: 85/15; Column temperature: 26 DEG C; Determined wavelength: 238nm; Flow velocity: 1.0m, L/min), obtain grey mold quinone A (
1, 30.6mg,
t r16.46min) and grey mold quinone B (
2, 11.8mg,
t r21.45min).
According to the UV spectrum (accompanying drawing 1 and 15) of grey mold quinone A and grey mold quinone B, optical rotational activity spectrum,
1h composes (accompanying drawing 2 and 16),
13c spectrum (accompanying drawing 3 and 17),
1h-
1hCOSY composes (accompanying drawing 4,5,18,19 and 20), hsqc spectrum (accompanying drawing 6,7,8 and 21), HMBC composes (accompanying drawing 9,10,11,12,13,22,23,24 and 25), NOESY composes (accompanying drawing 26) and high resolution mass spectrum (accompanying drawing 14 and 27), and the Structural Identification of grey mold quinone A and grey mold quinone B is new compound, and its main COSY, HMBC and NOE dependency is see accompanying drawing 28.
The physico-chemical property of grey mold quinone A: burgundy powder; Molecular formula C
27h
25nO
8; [
]
d 25+ 63.7 (
c0.50, DMSO); UV (MeOH):
240,327,443nm; High resolution mass spectrum (HRESIMS) is
m/z[M+Na]
+514.1457 (calculated value C
27h
25nNaO
8, 514.1478);
13c and
1hNMR signals assignment is in table one.
The physico-chemical property of grey mold quinone B: burgundy powder; Molecular formula C
28h
22o
8; [
]
d 25+ 51.9 (
c0.50, DMSO); UV (MeOH):
221,237,327,445nm; High resolution mass spectrum (HRESIMS) is
m/z[M+Na]
+509.1233 (calculated value C
28h
22naO
8, 509.1212);
13c and
1hNMR signals assignment is in table two.
Determine that described grey mold quinone A and grey mold quinone B is new compound, their chemical structural formula is:
。
the bioactivity research of grey mold quinone A and grey mold quinone B
5.1. the effect of grey mold quinone A and grey mold quinone B inhibition tumor cell propagation
Rat brain glioma C6 cell and human glioma U251 cell DMEM and 10%FBS substratum are cultivated in the incubator of 37 DEG C and 5% carbonic acid gas, and human glioma U87-MG and HSG-44 cell are cultivated in the incubator of 37 DEG C and 5% carbonic acid gas with MEM substratum and RPMI-1640 substratum respectively, be used for experimental study of the present invention through three generations's cultured cells.
Measure tumor cell survival by Sulforhodamine B (SRB) method, Zorubicin (Doxorubicin) contrasts for positive drug.Cell is inoculated in 96 orifice plates, adds grey mold quinone A or the grey mold quinone B of different concns after adherent 24h.With SRB dyeing after drug treating 72h, measure the absorb light angle value at 515nm place by microplate reader, detect the survival rate of tumour cell, calculate the IC that grey mold quinone A and grey mold quinone B suppresses glioma
50value.Experimental result shows: grey mold quinone A and grey mold quinone B significantly suppresses the propagation of glioma cell, its IC
50value is respectively 0.71-3.93
and 4.64-10.48
(table three, accompanying drawing 29 and accompanying drawing 30).
5.2. the effect of grey mold quinone A and grey mold quinone B inducing apoptosis of tumour cell
The apoptotic effect of people glioma U251 is induced to carry out quantitative analysis with the two staining analysis method of AnnexinV-FITC/PI to grey mold quinone A and grey mold quinone B.By glioma U251 cell grey mold quinone A (0.71
) or grey mold quinone B (4.65
) process 36 hours after, collect 1
10
6individual cell.Cell is dispersed in 100 again with after cold PBS buffer solution
containing 5
annexinV-FITC and 1
in the binding buffer liquid of 100 μ g/mlPI working fluids.Cell adds 400 after at room temperature hatching 15 minutes
binding buffer liquid, by its fluorescence of flow cytomery (excitation wavelength: 488nm; Emission wavelength: 530nm and 575nm).Experimental result shows: compare with control group U251 apoptosis (3.58%), grey mold quinone A and the rear 36h of grey mold quinone B process, Apoptosis of Human Glioma U 251 cell rate can be caused respectively to raise 38.76% and 36.67% (see accompanying drawing 31), in figure, the lower left corner is normal cell, the lower right corner is viable apoptotic cell, the upper right corner is non-viable apoptotic cell, and the upper left corner is non-viable non-apoptotic cell.
Above result shows: grey mold quinone A and grey mold quinone B significantly suppresses brain glioblastoma cell to be bred, and obviously induces brain glioblastoma cell apoptosis.So the related drugs prepared by grey mold quinone A and grey mold quinone B has application prospect in the treatment of cerebral glioma.
<110> Zhejiang University
<120> grey mold quinone A and B and preparation method thereof and medicinal use
<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 (StreptomycesgriseusP82SMLY)
<220>
<222>(1)…(1255)
<440>1
ACTGCGTCACCTTCGAAGCTCCCTCCCACAAGGGGTTGGGCCACCGGCTT50
CGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGG100
GAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGAC150
TTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTG200
AGATTCGCTCCGCCTCGCGGCATCGCAGCTCATTGTACCGGCCATTGTAG250
CACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCC300
ACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAGTCCCCATCACCCC350
GAAGGGCATGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTT400
AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTAT450
ACCGACCACAAGGGGGGCACCATCTCTGATGCTTTCCGGTATATGTCAAG500
CCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCTG550
CTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTAC600
TCCCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACCGACGACGTGGAA650
TGTCGCCAACACCTAGTTCCCAACGTTTACGGCGTGGACTACCAGGGTAT700
CTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAATGGCC750
CAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCA800
CCGCTACACCAGGAATTCCGATCTCCCCTACCACACTCTAGCTAGCCCGT850
ATCGAATGCAGACCCGGGGTTAAGCCCCGGGCTTTCACATCCGACGTGAC900
AAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGC950
CCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCCGGCGCTTCTTCT1000
GCAGGTACCGTCACTTTCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCC1050
GAAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCAT1100
TGTGCATATTCCCCACTGCTGCCTCCGTAGAGTCTGGTCGTGTCTTCAGT1150
TCCAGTGTGTGTCGCCCCTCCCTCAGCGCCTACCGTCGGTCGCCTTGTTA1200
GCATACCCCACACCAGCTGAATGCGCGGCTCATCCTTCCACCCGCCGGAG1250
CATCA1255
Claims (7)
1. a streptomyces griseus bacterial strain P82SMLY, is characterized in that: described streptomyces griseus bacterial strain is preserved in China typical culture collection center, and deposit number is CCTCCNO:M2015386, and Classification And Nomenclature is: streptomyces griseus P82SMLY(
streptomycesgriseusp82SMLY), preservation day: on June 19th, 2015.
2. a kind of streptomyces griseus bacterial strain P82SMLY according to claim 1, be is characterized in that, obtained by following steps separation and Culture:
(1) streptomyces griseus
streptomycesgriseusthe separation and Culture of P82SMLY
Get the dilution of air dried oceanic sediment strain cultivation liquid, concentration after dilution is 0.1-0.001g/mL, sample thief diluent evenly spreads in the culture dish containing solid medium, at ambient temperature after incubation time 7-15 days, different bacterium colonies is transferred to another respectively to be contained in the culture dish of solid medium, continue at ambient temperature to cultivate 7-15 days, finally well-grown single colony inoculation is placed on 4 DEG C of Refrigerator stores to slant medium cultivation for subsequent use;
Described oceanic sediment obtains from Zhoushan Of Zhejiang Province Area of The East China Sea; The described every 100mL of strain cultivation liquid consists of: 1.5 grams of glucose, 1.5 grams of glycerol, 1.5 grams of malt extracts, 2.5 grams of yeast extracts, 0.5 gram of casamino acids and 0.1 gram of calcium carbonate, or other liquid nutrient medium formed that Suitable strains P82SMLY grows; The sampling amount of described sample diluting liquid is 100-300
; Solid medium contained by described culture dish is bacteria Agr Bacto-agar substratum or other solid medium; Described slant medium is that Gao Shi synthesizes slant medium or other solid slant culture base; Described incubated at room temperature temperature is 20-30 DEG C;
(2) strain identification: the 16SrDNA sequence analysis method qualification generally used with current laboratory by the bacterial strain of above-mentioned steps (1) separation and Culture gained, be defined as streptomyces griseus, Classification And Nomenclature is
streptomycesgriseusp82SMLY, is deposited in China typical culture collection center, deposit number CCTCCNO:M2015386.
3. anticol matter tumor activity material grey mold quinone A (streptoanthraquinoneA) and grey mold quinone B (streptoanthraquinoneB), is characterized in that, described grey mold quinone A and grey mold quinone B has following chemical structural formula:
。
4. the preparation method of anticol matter tumor activity material grey mold quinone A and grey mold quinone B according to claim 3, be is characterized in that, realized by following steps:
(1) grey mold quinone A and grey mold quinone B producing strains streptomyces griseus
streptomycesgriseusthe preparation of P82SMLY zymocyte liquid
Get streptomyces griseus
streptomycesgriseusthe colony inoculation of P82SMLY is to containing in the large Erlenmeyer flask of liquid spawn culture medium, nutrient solution containing P82SMLY bacterial classification is rotated at ambient temperature jolting to cultivate after 5-15 days to prepare strain liquid, finally strain liquid is proceeded to the large Erlenmeyer flask containing liquid fermentation medium, rotate jolting at ambient temperature after fermentation culture 5-15 days, obtain the P82SMLY zymocyte liquid with anti-microbial activity;
The formula of the every 1000mL of described liquid spawn culture medium is: 20.0 grams of starch, 1.0 grams of saltpetre, 0.5 gram of dipotassium hydrogen phosphate, 0.5 gram of magnesium sulfate, 0.5 gram of sodium-chlor and 0.01 gram of ferric sulfate, and amount used is 150-300mL; Described liquid fermentation medium is liquid Gao Shi synthetic medium, and amount used is 250-500mL; Described large triangle culturing bottle is 500 or 1000mL; Described incubated at room temperature temperature is 20-30 DEG C; The speed of rotation of described rotation jolting is 160-200rpm;
(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 and obtains acetic acid ethyl ester extract, then octadecylsilane chemically bonded silica column chromatography for separation is used, use the methanol-eluted fractions of 70%, 85% and 100% respectively, obtain three components, component 3 continuation high performance liquid phase is separated, and obtains grey mold quinone A and grey mold quinone B;
The ODS consumption of described column chromatography and the sample size ratio of upper amount are 40-60g:1.0g; Described high performance liquid phase separation condition is: Agilent1260 high performance liquid chromatograph, Agilent1260DAD detector, AgilentZorbaxSB-C
18chromatographic column: 250
9.4mm, 5
, methanol/water ratio is 85/15 as moving phase, column temperature 26 DEG C, determined wavelength 238nm, and flow velocity is 1.0mL/min;
(3) Structural Identification of grey mold quinone A and grey mold quinone B
By the NMR spectrum of UV spectrum, a peacekeeping two dimension and the structure of high resolution mass spectrum data authentication grey mold quinone A and grey mold quinone B.
5. the application of grey mold quinone A according to claim 3 and grey mold quinone B in preparation treatment cerebral glioma medicine.
6. application according to claim 5, it is characterized in that, described medicine is that grey mold quinone A or grey mold quinone B activeconstituents are independent, or grey mold quinone A and grey mold quinone B share, or grey mold quinone A with grey mold quinone B with other medicines or together with effective constituent, the medicine formed with pharmaceutically acceptable carrier.
7. application according to claim 6, is characterized in that, the dosage form of described medicine is liquid preparation, solid preparation, capsule preparations, sustained release preparation, nanometer formulation.
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