CN105968067B - Valinomycins B and preparation and medical usage - Google Patents

Valinomycins B and preparation and medical usage Download PDF

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CN105968067B
CN105968067B CN201510908937.3A CN201510908937A CN105968067B CN 105968067 B CN105968067 B CN 105968067B CN 201510908937 A CN201510908937 A CN 201510908937A CN 105968067 B CN105968067 B CN 105968067B
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valinomycins
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glioma
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张治针
叶雪威
连晓媛
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Zhejiang University ZJU
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Abstract

The present invention provides a kind of compound valinomycins B with anticol matter tumor activity, there is provided is separately cultured from ooze to active material producing strains streptomyces parvus, and therefrom prepares active material valinomycins B.The streptomyces parvus is preserved in China typical culture collection center, and Classification And Nomenclature is streptomyces parvus P11 23B, (Streptomyces parvulus P11 23B) deposit number CCTCC NO：M 2015675, preservation day 2015.11.12.Valinomycins B, which has, significantly inhibits a variety of brain glioblastoma cell propagation, obvious retardance glioma cell cycle in G0/G1Phase, reduce glioma cell different metabolic approach multiple key enzymes protein expression, there is unique antitumor action.Therefore, valinomycins B can prepare the application in treating glioma medicine.Valinomycins B chemical structural formulas are：

Description

Valinomycins B and preparation and medical usage
Technical field
The invention belongs to field of medicaments, is related to from marine actinomycete streptomyces parvus (Streptomyces parvulus P11- The method and the compound that active compound for anti tumor valinomycins B (Valinomycin B) is prepared in 23B) are preparing treatment Application in terms of glioma medicine.
Background technology
Glioma is brain tumor most common in brainpan and most pernicious, accounts for the 70% of all malignant brain tumors.Defend in the world Raw tissue statistics shows：Glioblastoma is the 2nd cause of death of less than 34 years old tumor patient, is 35~54 years old patient The 3rd cause of death, seriously endanger the health and life of the mankind.For glioma since location is special, operating difficulty is big And be not easy tumour complete resection, so it is particularly important that the treatment of radiotherapy and medicine to glioma.But anticol matter at present Tumor medicine famine, Temozolomide are unique selectable medicines for being individually used for Treatment for Glioma.Moreover, including for not azoles The effect of existing treatment colloid tumor medicine including amine, is limited, has wretched insufficiency, outstanding behaviours is more:(1) it is mostly chemicals, poison Side effect is big；(2) serious drug resistance of the tumour cell to medicine；(3) blood-brain barrier hinders medicine arrival intracerebral and plays its treatment Effect.Therefore, clinically need to overcome disadvantages described above, the new anticol matter of better efficacy, security higher and mechanism of action uniqueness Tumor medicine.
Numerous studies prove：Excessive glycolysis (Glycolysis), vigorous glutamine decompose (Glutaminolysis) and lipid synthesis (Lipogenesis) is glioma cell metabolism dashing forward different from normal cell metabolism Go out feature.Glioma cell absorbs a large amount of glucose from microenvironment and is specifically relied on by glioma cell and high in glycolysis link Multiple key enzymes of expression include Hexokinase 2 (HK2), phosphofructokinase/fructose 2,6- diphosphatases (PFKFB3), acetone Acid kinase M2 (PKM2) and lactic dehydrogenase 5 (LDH5) etc. participate in producing a large amount of intermediate products and end-product lactic acid jointly, these Intermediate product is the starting material of tumour cell synthesising biological macromolecular, and Lactic Acid Secretion is thin to extracellularly can inhibit immunity of organism Born of the same parents can promote tumour cell to spread, the glycolysis key enzyme HK2 of height expression can also improve tumour the Scavenging activity of tumour cell Resistance of the cell to radiotherapy and Temozolomide chemotherapy.Meanwhile the vigorous glutamine decomposition that glutaminase (GLS) is leading, Substantial amounts of nitrogen source is provided for the synthesis of biomolecule, and the active lipid conjunction that the fatty acid synthetase (FASN) that height is expressed is leading Into the generation for being conducive to cell membrane.It can be appreciated that exactly tumour cell is highly divided greatly using its metabolic intermediate synthesising biological The ability of sub- DNA, RNA, protein and biomembrane promote it quickly unrestrictedly to breed, and modulate tumor is metabolized the pass of different links Key enzyme can effectively suppress the propagation of tumour cell.Therefore, the multiple target effect of the different link key enzymes of glioma metabolism is targeted Medicine may have more preferable antitumor curative effect.Valinomycins B (Valinomycin B) is from marine bacteria streptomyces parvus P11- Isolated noval chemical compound in 23B (Streptomyces parvulus P11-23B), the compound is to a variety of different colloids The propagation of oncocyte has significant inhibitory action, can obviously reduce glioma cell glycolysis, glutamine decomposes and lipid The protein expression level of several key enzymes in route of synthesis, has unique Anticancer Effect and Mechanism, therefore, valinomycins B exists Have a good application prospect in terms of preparing anticol matter tumor medicine.
The content of the invention
It is an object of the present invention to provide a kind of compound valinomycins B with anticol matter tumor activity (Valinomycin B, compound 1), the valinomycins B are a noval chemical compound, its chemical structural formula is：
It is a further object to provide the preparation method of valinomycins B (Valinomycin B), pass through following step It is rapid to realize：
(1) preparation of streptomyces parvus Streptomyces parvulus P11-23B zymocyte liquids
The colony inoculation of streptomycete Streptomyces parvulus P11-23B is taken to containing a certain amount of liquid spawn In the big conical flask of culture medium, by the nutrient solution containing P11-23B strains, shaken cultivation is after a certain period of time at ambient temperature To prepare strain liquid.Strain liquid is finally transferred to the big conical flask containing a certain amount of liquid fermentation medium, in room temperature bar Oscillation and fermentation cultivation after a certain period of time, obtains the zymocyte liquid of P11-23B with antitumor activity under part.
The liquid spawn culture medium and liquid fermentation medium is liquid Gao Shi culture mediums；The dosage is 100-200mL；The big triangle blake bottle is 250 or 500mL；The incubated at room temperature temperature is 20~30 DEG C；It is described to shake The rotating speed swung is 160-180rpm；The incubation time is 5~15 days.
(2) extraction separation and purification of valinomycins B (Valinomycin B)
The zymocyte liquid of bacterial strain P11-23B is extracted with ethyl acetate to obtain acetic acid ethyl ester extract, acetic acid ethyl ester extract With octadecylsilane chemically bonded silica (ODS) column chromatography for separation, eluted respectively with 70% and 100% methanol, 100% methanol The residue through being concentrated under reduced pressure to give is separated with HPLC after eluent merges, and obtains pure compound valinomycins B (Valinomycin B)。
The ODS dosages of the column chromatography and the sample size ratio of upper amount are 40~60g:1.0g；The efficient liquid phase point It is from condition:1260 high performance liquid chromatographs of Agilent, Agilent 1260DAD detectors, Agilent Zorbax SB- C18Chromatographic column (250 × 9.4mm, 5 μm), 100% methanol are mobile phase, 26 DEG C, Detection wavelength 210nm of column temperature, and flow velocity is 1.0mL/min。
(3) Structural Identification of valinomycins B (Valinomycin B)
The structure of valinomycins B (Valinomycin B) is NMR spectra, the high-resolution according to their peacekeeping two dimension What the methods of mass spectrometric data and chemical degradation, was identified.
Third object of the present invention is to provide a kind of streptomyces parvus P11-23B, and the bacterial strain is preserved in Chinese Typical Representative Culture collection, Classification And Nomenclature are streptomyces parvus P11-23B (Streptomyces parvulus P11-23B), preservation Numbering CCTCC NO：M 2015675, preservation day 2015.11.12, preservation address：Wuhan, China.
Fourth object of the present invention is to provide the preparation method of the streptomyces parvus P11-23B, is a kind of from ooze The method of middle bacterial strain P11-23B of the separation with anticol matter tumor activity, is separately cultured by following steps to obtain：
(1) streptomyces parvus (Streptomyces parvulus P11-23B) is separately cultured
Take air dried ooze to be diluted to certain concentration with seawater, take a certain amount of sample diluting liquid to evenly spread to In culture dish containing solid medium, cultivate at ambient temperature after a certain period of time, different bacterium colonies is transferred to separately respectively In one culture dish containing solid medium, continue to cultivate certain time at ambient temperature.Finally will be well-grown single Bacterium colony (P11-23B) is inoculated into 4 DEG C of refrigerators of slant medium culture postposition and saves backup.
The ooze and seawater is obtained from Zhoushan Of Zhejiang Province Area of The East China Sea；The concentration of the sample diluting liquid for 1 × 10-6~1 × 10-4g/mL；The sampling amount of the sample diluting liquid is 100~300 μ L；Solid training contained by the culture dish It is Gao Shi agar (Gause ' s agar) culture medium or other solid mediums to support base；The slant medium is Gao Shi agar Culture medium or other solid slope culture mediums；The incubated at room temperature temperature is 20~30 DEG C；The incubation time is 7~15 My god.
(2) strain idenfication of streptomyces parvus (Streptomyces parvulus P11-23B)
Above-mentioned steps (1) are separately cultured the 16S rDNA sequence analyses that obtained bacterial strain is generally used with current laboratory The species of method identification bacterial strain P11-23B, is determined as streptomyces parvus, Classification And Nomenclature is Streptomyces parvulus P11- 23B, by China typical culture collection center-Wuhan center preservation, deposit number CCTCC NO：M 2015675.
The 5th purpose of the present invention is to provide valinomycins B and is preparing the application in treating glioma medicine.It is described Valinomycins B can significantly inhibit the propagation of a variety of glioma cells, the retardance glioma cell cycle in the G0/G1 phases, reduces glue The protein expression of multiple key enzymes, has uniqueness in the glycolysis of matter oncocyte, glutamine decomposition and lipid route of synthesis Function of tumor mechanism.
The medicine is independent for valinomycins B activity component, or valinomycins B and other medicines or with active ingredient one Rise, the medicine with pharmaceutically acceptable carrier composition.The dosage form of the medicine is：Liquid preparation, solid pharmaceutical preparation, capsule Preparation, sustained release preparation, nanometer formulation.
The present invention is separately cultured from ooze to active material producing strains streptomyces parvus Streptomyces parvulus P11-23B, and therefrom it is found that new active material valinomycins B (Valinomycin B).Valinomycins B significantly inhibits more Kind brain glioblastoma cell propagation, hence it is evident that the retardance glioma cell cycle is in G0/G1Phase, reduces glioma cell different metabolic approach Multiple key enzymes protein expression, there is unique Anticancer Effect and Mechanism.Therefore, valinomycins B is preparing treatment There is application prospect in terms of glioma medicine.
Brief description of the drawings
Fig. 1 is the bacterium colony figure of streptomyces parvus Streptomyces parvulus P11-23B.
Fig. 2-6 is the hydrogen spectrum of valinomycins B (Valinomycin B).
Fig. 7-11 is the carbon spectrum of valinomycins B (Valinomycin B).
Figure 12 is valinomycins B (Valinomycin B)1H-1H COSY are composed.
Figure 13-16 is the hsqc spectrum of valinomycins B (Valinomycin B).
Figure 17-23 is the HMBC spectrums of valinomycins B (Valinomycin B).
Figure 24 is the high resolution mass spectrum of valinomycins B (Valinomycin B).
Figure 25 is valinomycins B (Valinomycin B) to the inhibitory action of glioma.
Figure 26 is that valinomycins B (Valinomycin B) blocks the glioma U87-MG cell cycles in the G0/G1 phases.
Figure 27 is that valinomycins B (Valinomycin B) blocks the glioma U251 cell cycles in the G0/G1 phases.
Figure 28 is that valinomycins B (Valinomycin B) reduces glioma U87-MG cell different metabolic pathway key enzymes Protein expression level (CON：U87-MG cell controls groups；1：Valinomycins B treatment groups；DMSO：Dimethyl sulfoxide solvent compares Group；HK2:Hexokinase：PFKFB3:Phosphofructokinase/fructose 2,6- diphosphatases；PKM2:Pyruvate kinase M2；GLS:Paddy Transglutaminase；FASN：Fatty acid synthetase；β-ACTIN:Beta-actin, internal reference).
Embodiment
The present invention is described in further detail below in conjunction with drawings and examples.It is real but the present invention is not restricted to these Apply example.
1. streptomyces parvus P11-23B of embodiment (Streptomyces parvulus P11-23B's) is separately cultured
Air dried 1 gram of ooze is taken to be diluted to concentration with seawater as 1 × 10-6The sample diluting liquid of g/mL, takes 200 μ L's Sample diluting liquid is evenly spread in the culture dish containing Gao Shi agar (Gause ' s agar) solid medium, in 28 DEG C of conditions After lower culture 7 days, different bacterium colonies is transferred in another culture dish containing Gao Shi Solid agar cultures respectively, at 28 DEG C Under the conditions of continue culture 7 days.Well-grown single bacterium colony (P11-23B) is finally inoculated into the training of Gao Shi agar slant culture-mediums 4 DEG C of refrigerators of postposition are supported to save backup.
The strain idenfication of 2. streptomyces parvus P11-23B of embodiment (Streptomyces parvulus P11-23B)
The species of bacterial strain P11-23B 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 It is:TACGGYTACCTTGTTACGACTT and AGAGTTTGATCMTGGCTCAG；
Marker：DL500
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
B.PCR reaction conditions
10 DEG C of ∞,
C. electrophoresis detection,
D. it is sequenced：Glue purification sequencing is cut,
E. analysis result：Splice sequence.
2.3 experimental result
Spliced sequence is：
AACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCT GGAAACGGGGTCTAATACCGGATACTGACCTTCACGGGCATCTGTGAAGGTCGAAAGCTCCGGCGGTGCAGGATGAG CCCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCG GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCC TGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGAC GGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAA TTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGT CGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGG AACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTAAC GCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGC GGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGTCCAGAGATGG GCGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG CAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCA ACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGG TACAATGAGCTGCGATACCGCAAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCG ACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCTCAACCC。
16S rDNA sequences achieved above compared with the NCBI GenBank databases of U.S. NIH, itself the result shows that：Bacterium The 16S rDNA sequences and the 16S of the Streptomyces parvulus strain PFS10 in GenBank storehouses of strain P11-23B RDNA sequences have 99% similitude (accession number:KJ789323.1).Therefore, the marine bacteria strain P11-23B that the present invention is obtained It is set to streptomyces parvus P11-23B (Streptomyces parvulus P11-23B) (attached drawing 1).Streptomyces parvus P11-23B (Streptomyces parvulus P11-23B) bacterial strain by China typical culture collection center-Wuhan center preservation, is protected Hide numbering CCTCC NO：M 2015675.
The preparation of 3. streptomyces parvus P11-23B of embodiment (Streptomyces parvulus P11-23B) zymocyte liquid
The colony inoculation of streptomycete Streptomyces parvulus P11-23B is taken to be trained to containing 200mL liquid Gao Shi In the 500mL conical flasks for supporting base, the nutrient solution containing P11-23B strains is rotated to (180rpm) shaking training under the conditions of 28 DEG C Strain liquid is obtained after supporting 7 days.5mL strain liquids are transferred in the 500mL conical flasks containing 200mL liquid Gao Shi culture mediums, (180rpm) shaking culture 7 days is rotated under the conditions of 28 DEG C, obtains the zymocyte liquid of P11-23B with antitumor activity.
The extraction separation and purification of 4. valinomycins B of embodiment (Valinomycin B)
The zymocyte liquid (50.0 liters) that Example 3 obtains is extracted with ethyl acetate to obtain acetic acid ethyl ester extract (7.56 Gram), acetic acid ethyl ester extract octadecylsilane chemically bonded silica (ODS, 500 grams) column chromatography for separation, respectively with 2000mL's 70% and 100% methanol elution, (1.38 grams) use of residue through being concentrated under reduced pressure to give after 100% meoh eluate merging HPLC separates (instrument：Agilent 1260；Chromatographic column：Agilent Zorbax SB-C18, 250 × 9.4mm, 5 μm；Mobile phase: 100% methanol；Column temperature：26℃；Detection wavelength：210nm；Flow velocity：1.0mL/min), valinomycins B (156mg, t are obtainedR 18.34min)。
Valinomycins B is colourless powder；Molecular formula C53H88N6O18；[α]D 25+36.8.7(c 0.36,MeOH)；IR(KBr) 2967,2936,2873,1743,1655,1193,1094,1027mm；High resolution mass spectrum (HRESIMS) is m/z [M-H]- 1095.6051 (calculated value C53H87N6O181095.6077).Handle acidic amino acid column and gas chromatographic analysis, and with standard items pair According to, valinomycins B acid hydrolysis products be accredited as D-Val (D-Val), Valine (L-Val), Pfansteihl (L-Lac), D- Alpha-hydroxies isovaleric acid (D-Hiv), D- alpha-hydroxybutyric acids (D-Hbu).
According to valinomycins B's1H spectrums (attached drawing 2-6),13C spectrums (attached drawing 7-11),1H-1H COSY spectrums (attached drawing 12), HSQC Compose (attached drawing 13-16), HMBC spectrums (attached drawing 17-23), high resolution mass spectrum figure (attached drawing 24) and chemical degradation, the knot of valinomycins B Structure is accredited as noval chemical compound, its13C and1H NMR signals ownership is shown in Table one.
Table one, the carbon of valinomycins B and hydrogen signal ownership (in CDCl3)
The activity research of 5. valinomycins B of embodiment
5.1. valinomycins B suppresses 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 SHG-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 used for the present invention Experimental study.
Tumor cell survival is measured with Sulforhodamine B (SRB) method, adriamycin (Doxorubicin) is used for positive drug Control.Cell inoculation adds the valinomycins B of various concentrations in 96 orifice plates after adherent 24h.Contaminated after drug-treated 72h with SRB Color, with the absorbance value at microplate reader measure 515nm, detects the survival rate of tumour cell, calculates valinomycins B and suppress colloid The IC of tumor cell proliferation50Value.Test result indicates that：Valinomycins B significantly inhibits the propagation of glioma cell, its IC50Value difference For 0.25-0.41 μM (table two, attached drawing 25).
Table two, valinomycins B suppress the effect (IC of glioma50:μM)
5.2. valinomycins B blocks the glioma cell cycle in G0/G1Phase
After being dyed with propidium iodide (PI) DNA, flow cytometry analysis valinomycins B is thin to glioma U87-MG and U251 The influence in intracellular growth cycle.By glioma U87-MG and U251 cell respectively with (0.8 μM) of valinomycins B processing 12h, 24h, 48h or with after 72h, collect cell and after mix with 70% ethanol of frost it is overnight under the conditions of 4 DEG C.Mixed liquor after overnight Through centrifuging (1900rpm, 7 minutes) isolated cells rinsed with PBS twice.Cell is dispersed in containing RNase A's again In PBS, hatch 30 minutes at 37 DEG C, finally dyed 30 minutes at 4 DEG C of dark with propidium iodide (PI).With FACScan streamings Changes of the Cytometric Analysis valinomycins B to U87-MG the and U251 cell cycles.Experimental result is shown：(1) compared with the control group, After valinomycins B (0.8 μM) processing glioma U87-MG cells 12h, 24h and 48h, the G of cell cycle0/G1Phase cell proportion point 40.92%, 32.54% and 31.30% (table three, attached drawing 26) is not added.
The influence of table three, valinomycins B to the glioma U87-MG cell cycles
(2) compared with the control group, (0.8 μM) processing glioma U87-MG cells 12h, 24h, 48h and 72h of valinomycins B Afterwards, the G of cell cycle0/G1Phase cell proportion adds 40.92%, 32.54% and 31.30% (table four, attached drawing 27) respectively.
The influence of table four, valinomycins B to the glioma U251 cell cycles
It these results suggest that, valinomycins B retardances are in the glioma cell cycle in G0/G1Phase.
5.3. influences of the valinomycins B to glioma cell different metabolic pathway key enzyme
The preparation of protein sample：People's glioma U87-MG cells are with MEM and 10%FBS culture mediums in 37 DEG C and 5% dioxy Change and cultivated in the incubator of carbon, the cell by three generations's culture is used for the experimental study of the present invention.Cell (1.5 × 107) inoculation Added in 10 centimetres of culture dish, after adherent 24h (5.0 μM or 10.0 μM) of valinomycins B co-culture 48 it is small when after, first use ice Cold PBS buffer is washed twice, and rear ice-cold lysis buffer (200 μ L) cracks 15 minutes.Lysate is through 4 DEG C of low-temperature and high-speeds (11200rpm) is centrifuged, and supernatant is protein sample.
The measure of protein content：Use the protein content of each protein sample of BCA kit measurements.By seminal plasma fructose detection kit A and B are with 50：1 ratio is made into working reagent liquid, and standard items BCA dilutions are made into various concentrations with bovine serum albumin(BSA) (BSA) BCA standard solution.10 μ L standard solution or protein sample liquid and 200 μ L working reagents liquid are taken at 37 DEG C to hatch 30 points after mixing Clock artemia hatching solutions microplate reader measures trap in 562nm wavelength.Using BCA amounts as abscissa, trap draws standard for ordinate Curve, calculates regression equation.From the protein content of each protein sample liquid of regression equation calculation.
Western Blotting：10% dodecyl sodium sulfate polyacrylamide of each sample containing equal protein (15 μ g) Amine gel electrophoresis (SDS-PAGE) separates, and gel electrophoresis protein graphical spectrum is gone on polyvinylidene fluoride film (PVDF), and pvdf membrane is used The 0.1%TBST of also 5% degreasing milk close at room temperature 2 it is small when.Pvdf membrane after closing first with HK2, PFKFB3, The primary antibody of PKM2, FASN and GLS enzyme is incubated overnight at 4 DEG C, and film is washed with TBST；It is small in incubation at room temperature 2 with the secondary antibody of HRP marks afterwards When.After TBST washes film three times, immunoreactivity is detected with enhanced chemiluminescence reagent, is then developed, is washed, is fixed, washing, dries in the air It is dry, observation experiment result.Compareed using beta-actin (β-ACTIN) as internal reference.
Test result indicates that：Compared with negative control group (without the U87-MG cells of drug-treated), valinomycins B (10.0 μM) significantly reduce the protein level table of the key enzyme HK2, PFKFB3, FASN and GLS of tumour cell different metabolic approach Up to (attached drawing 28).This result is prompted, and valinomycins B may be by influencing glioma glycolysis, glutamine decomposition and lipid The key enzyme of synthesis and produce its antitumor activity, there is unique Mutiple Targets Anticancer Effect and Mechanism.
In conclusion present invention demonstrates that valinomycins B significantly inhibits the propagation of a variety of glioma cells, blocks tumor cells Cycle is in G0/G1Phase, significantly reduces the protein expression of multiple key enzymes during glioma cell different metabolic, has only Special Anticancer Effect and Mechanism, so, by related drugs prepared by valinomycins B before there is application in terms for the treatment of glioma Scape.

Claims (1)

1. a kind of preparation method of anticol matter tumor activity material valinomycins B, it is characterised in that realized by following steps：
(1) preparation of streptomyces parvus P11-23B zymocyte liquids：
The colony inoculation of streptomyces parvus P11-23B is taken into the big conical flask containing a certain amount of liquid spawn culture medium, will Shaken cultivation is after a certain period of time to prepare strain liquid at ambient temperature for nutrient solution containing P11-23B strains, finally by strain Liquid is transferred to the big conical flask containing a certain amount of liquid fermentation medium, at ambient temperature oscillation and fermentation cultivation certain time Afterwards, the zymocyte liquid with the P11-23B of antitumor activity is obtained；
The liquid spawn culture medium and liquid fermentation medium is liquid Gao Shi culture mediums；The dosage is 100- 200mL；The big triangle blake bottle is 250 or 500mL；The incubated at room temperature temperature is 20~30 DEG C；The vibration Rotating speed be 160-180rpm；The incubation time is 5~15 days；
(2) extraction separation and purification of valinomycins B
The zymocyte liquid of bacterial strain P11-23B is extracted with ethyl acetate to obtain acetic acid ethyl ester extract, and acetic acid ethyl ester extract is with ten Eight alkyl silane bonded silica gel column chromatography for separation, are eluted with 70% and 100% methanol respectively, and 100% meoh eluate closes The residue through being concentrated under reduced pressure to give is separated with HPLC after and, obtains pure compound valinomycins B；
The octadecylsilane chemically bonded silica dosage of the column chromatography and the sample size ratio of upper amount are 40~60g：1.0g；It is described Efficient liquid phase separation condition be:1260 high performance liquid chromatographs of Agilent, 1260 DAD detectors of Agilent, Agilent Zorbax SB-C18Chromatographic column 250 × 9.4mm, 5 μm, 100% methanol is mobile phase, 26 DEG C of column temperature, Detection wavelength 210nm, flow velocity 1.0mL/min；
(3) Structural Identification of valinomycins B：
The knot of valinomycins B is identified according to the NMR spectra of peacekeeping two dimension, high resolution mass spectrum data and chemical degradation method Structure.
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