CN109081863A - The isolation and identification method of anti-cancer active matter D actinomycin D FGR - Google Patents
The isolation and identification method of anti-cancer active matter D actinomycin D FGR Download PDFInfo
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- CN109081863A CN109081863A CN201810866167.4A CN201810866167A CN109081863A CN 109081863 A CN109081863 A CN 109081863A CN 201810866167 A CN201810866167 A CN 201810866167A CN 109081863 A CN109081863 A CN 109081863A
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- fgr
- actinomycin
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- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Chemical compound CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 title claims abstract description 475
- 108010092160 Dactinomycin Proteins 0.000 title claims abstract description 168
- 229960000640 dactinomycin Drugs 0.000 title claims abstract description 168
- 238000000034 method Methods 0.000 title claims abstract description 32
- 230000001093 anti-cancer Effects 0.000 title claims abstract description 26
- 238000002955 isolation Methods 0.000 title claims abstract description 18
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 27
- 238000001228 spectrum Methods 0.000 claims abstract description 21
- 239000000126 substance Substances 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 239000007791 liquid phase Substances 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 105
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 32
- 238000010521 absorption reaction Methods 0.000 claims description 21
- 241000186046 Actinomyces Species 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 11
- 229930000044 secondary metabolite Natural products 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 241001655322 Streptomycetales Species 0.000 claims description 6
- 244000309464 bull Species 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 241000220324 Pyrus Species 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 235000021017 pears Nutrition 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 241000970845 Streptomyces tauricus Species 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 229910052603 melanterite Inorganic materials 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 235000010333 potassium nitrate Nutrition 0.000 claims description 2
- 238000012797 qualification Methods 0.000 claims description 2
- 230000000717 retained effect Effects 0.000 claims description 2
- 229910052564 epsomite Inorganic materials 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- 239000013049 sediment Substances 0.000 claims 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 abstract description 28
- 238000010828 elution Methods 0.000 abstract description 5
- 239000004615 ingredient Substances 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 31
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 12
- 238000004090 dissolution Methods 0.000 description 9
- 229910021645 metal ion Inorganic materials 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 230000031700 light absorption Effects 0.000 description 8
- 239000003755 preservative agent Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 239000007800 oxidant agent Substances 0.000 description 7
- 230000001590 oxidative effect Effects 0.000 description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 239000003638 chemical reducing agent Substances 0.000 description 6
- 235000019253 formic acid Nutrition 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 244000061458 Solanum melongena Species 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000004566 IR spectroscopy Methods 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- QSKWJTXWJJOJFP-UHFFFAOYSA-N chloroform;ethoxyethane Chemical compound ClC(Cl)Cl.CCOCC QSKWJTXWJJOJFP-UHFFFAOYSA-N 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Landscapes
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the isolation and identification methods of anti-cancer active matter D actinomycin D FGR, comprising the following steps: 1) D actinomycin D FGR is carried out all-wave length by ultraviolet specrophotometer UV and sweep spectrum detection；2) D actinomycin D FGR is further identified with high-efficient liquid phase chromatogram HPLC again；3) it is separated after identifying using high-efficient liquid phase chromatogram HPLC.The invention has the benefit that the present invention, which carries out ultraviolet specrophotometer to anti-cancer active matter D actinomycin D FGR first, sweeps spectrum identification, the results showed that D actinomycin D FGR of the present invention and actinomycin D are not same substances；Then further D actinomycin D FGR is identified with high performance liquid chromatography, shows that D actinomycin D FGR and actinomycin D are not same substances again；Then high performance liquid chromatography is used again, carries out gradient elution and initial gross separation, the ingredient that at least 11 kinds or more of this anti-cancer active matter are carried out to D actinomycin D FGR, and contain the homologue of actinomycin D, content 0.015mg/mL in D actinomycin D FGR.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular to the separation identification side of anti-cancer active matter D actinomycin D FGR
Method.
Background technique
Malignant tumour is the main lethal disease in one, the whole world, according to World Health Organization, the whole world in 2012 because
Mortality of malignant tumors person-time up to 8,200,000, it is contemplated that Malignant Tumor Case will continue rapidly to increase, by cumulative year after year to 2025
19000000 people, 2035 up to 24,000,000 people.Tumour seriously threatens human life and health, and existing most chemicals are in treatment disease
While sick, not only killing tumor cell, normal cell also receive serious injury, thus make patient produce it is serious not
Good reaction.Therefore, it is extremely urgent to seek a kind of anti-tumor drug efficiently, less toxic, it will help improve drug safety and have
Effect property.
Actinomyces are the feature-rich Pseudomonas of a kind of secondary metabolite, and secondary metabolite can be used as pigment, antibiosis
Element etc., actinomyces or a kind of anticancer drug with development and utilization potential quality, add in life science, food service industry, feed
The industries such as agent, medical science and chemical engineering industry are added to have potential application.But yield fermented, extracted etc. it is each not really
Determine the restriction of factor, and can not mass production.
Actinomyces secondary metabolite isolates and purifies process, is usually directed to separation and structure elucidation two large divisions.Currently, point
It is most commonly used from technical application, specifically include that ion-exchange chromatography, macroreticular resin absorbing method, aqueous two phase extraction technique, film
Isolation technics etc..Silica gel column chromatography, gel filtration chromatography, high performance liquid chromatography etc.；And structure elucidation is most typical no more than ultraviolet
The big wave spectrum of spectrum, infrared spectroscopy, mass spectrum and nuclear magnetic resoance spectrum etc. four can achieve test substance molecular structure level, thus
It is parsed and is identified.
Summary of the invention
The purpose of the present invention is to above-mentioned defects in the prior art, provide anti-cancer active matter D actinomycin D
The isolation and identification method of FGR.
The present invention from actinomyces secondary metabolite in order to obtain some new active ingredients, with actinomyces secondary
Metabolite antitumaous effect is purport, designs further separation and purification experiment to fermentation crude product, organic solvent is taken to extract
It follows the example of, ultraviolet spectrophotometer method (UV) and high performance liquid chromatography (HPLC).Select a variety of organic solvent detection D actinomycin D
Then the dissolubility of FGR detects pH, temperature, illumination (visible light and ultraviolet light), metal ion, preservative, oxidant and reduction
Influence of the agent to D actinomycin D FGR stability.
To achieve the goals above, technical solution provided by the invention are as follows: the separation of anti-cancer active matter D actinomycin D FGR
Identification method, comprising the following steps:
1) D actinomycin D FGR is subjected to all-wave length by ultraviolet specrophotometer UV and sweeps spectrum detection；
2) D actinomycin D FGR is further identified with high-efficient liquid phase chromatogram HPLC again；
3) it is separated after identifying using high-efficient liquid phase chromatogram HPLC；
Further, the isolation and identification method of above-mentioned anti-cancer active matter D actinomycin D FGR, the D actinomycin D FGR
It is the secondary metabolite of bull streptomycete FGR-015, isolates and purifies institute to expand medium liquid fermentation actinomyces FGR-015
?；
The actinomyces are streptomycete, are named as bull streptomycete (Streptomyces tauricus) FGR-015, are protected
Hiding unit is China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica, postcode 100101), the preservation time is on June 14th, 2018, and deposit number is
CGMCC No.15942；
The D actinomycin D FGR is bull actinomyces FGR-015 secondary metabolite, is put with expanding medium liquid fermentation
Line bacterium FGR-015 isolates and purifies gained；
The composition for expanding culture medium are as follows: soluble starch 20g, KNO3 0.1g, K2HPO40.05g, MgSO4·
7H2O 0.05g, NaCl 0.05g, FeSO4·7H2O 0.001g, distilled water 1000mL；PH value is adjusted to 7.1；
The expansion fermentation process are as follows: inoculum concentration 12.61%, bottling amount are 48.65%, shaking speed 150r/
Min, shaking table temperature are 28 DEG C, fermentation time 7.5d；
The isolation and purification method, comprising the following steps: centrifugal rotational speed 10000r/min, 4 DEG C of centrifugation 20min, it is heavy to abandon
Starch is extracted by extractant of ethyl acetate, and 30 DEG C of water bath sonicator 30min are then moved into pears type separatory funnel, stands 2h；
Using ethyl acetate as extractant, solid-liquid ratio 1:5, ultrasonic temperature is 44 DEG C, extraction time 16h；It is extracted with ethyl acetate 3
It is secondary；Separate organic phase, until solution to be measured be it is colourless, organic phase is retained and is passed through 60 DEG C of rotary evaporations, -20 DEG C icings and -
D actinomycin D FGR finished product is made in 80 DEG C of freeze-drying 3d.
Further, the isolation and identification method of above-mentioned anti-cancer active matter D actinomycin D FGR in the step 1), is put
It is maximum absorption wavelength 210nm that line rhzomorph FGR ultraviolet specrophotometer, which sweeps spectrum result,.
Further, the isolation and identification method of above-mentioned anti-cancer active matter D actinomycin D FGR in the step 1), is put
Line rhzomorph FGR ultraviolet specrophotometer sweeps spectrum method particularly includes: weighs 10mg D actinomycin D FGR with methanol and is dissolved as 10mg/
The solution of mL does blank control with methanol, and all-wave length is carried out on ultraviolet specrophotometer and sweeps spectrum, i.e. 190~1000nm sweeps spectrum,
Detect the maximum absorption wavelength of D actinomycin D FGR.
Further, the isolation and identification method of above-mentioned anti-cancer active matter D actinomycin D FGR in the step 2), is put
The high-efficient liquid phase chromatogram HPLC qualification result of line rhzomorph FGR is the substance containing 11 kinds or more.
Further, the isolation and identification method of above-mentioned anti-cancer active matter D actinomycin D FGR is high in the step 2)
The concrete operations of effect liquid phase chromatogram method are as follows: the D actinomycin D FGR of 10mg is dissolved as the solution of 10mg/mL with methanol；Chromatographically pure
Mobile phase methanol and pure water filter 7~10 times to complete free from admixture with vacuum pump respectively, and then ultrasound 30min, stands room temperature
It is spare；HPLC testing conditions are as follows: mobile phase is methanol: water=75:25, flow velocity 1mL/min elute 1h, and sample volume is 10 μ L, color
Composing column specification is PursuitXRs C18 (250mm × 10mm), and UV detector Detection wavelength is 252nm.
The present invention relates to the obtained actinomyces FGR-217 of screening in No. 1 culture medium growth course of solid Gao Shi, cell age by
Gradually mature, it is finally aubergine that the mode of appearance of bacterium colony, which gradually becomes pink, red by initial white, or even diffusion is whole
A culture medium.D actinomycin D FGR (temporary designations of actinomyces FGR-217 secondary metabolite) is purplish red coloured particles, is had certain
Viscosity, therefore can determine, haematochrome is generated in actinomyces FGR-217 metabolic process, and natural red colouring matter has extensive and valuable
Application value.In addition, experiment proves that, the D actinomycin D FGR is not only to some such as Escherichia coli, Pseudomonas aeruginosa, golden yellow Portugal
Grape coccus, Diplococcus pneumopniae pathogenic bacteria have certain inhibitory effect, still have certain lethality to Several Kinds of Malignancy,
Therefore, it identifies D actinomycin D FGR and just seems especially urgent to the research of its physicochemical property.
The invention has the benefit that the invention discloses the initial gross separations of anti-cancer active matter D actinomycin D FGR a kind of
The method and its physicochemical property of identification are explored.Ultraviolet specrophotometer is carried out to the anti-cancer active matter D actinomycin D FGR first
Sweep spectrum identification, result be a length of 210nm of maximum absorption wave, and a length of 252nm of object of suspicion actinomycin D maximum absorption wave and
428nm shows that D actinomycin D FGR of the present invention and actinomycin D are not same substances；Then with high performance liquid chromatography into
One step identifies D actinomycin D FGR, the D actinomycin D FGR solution appearance time of 3 kinds of formic acid, DMSO and methanol different solvents
In 2.0min, and actinomycin D appearance time is 10.589min, shows D actinomycin D FGR and actinomycin D again not
It is same substance；Then high performance liquid chromatography is used again, carries out gradient elution and D actinomycin D FGR is tentatively divided
From, it is known that, the ingredient that at least 11 kinds or more of this anti-cancer active matter, but have a peak in 11.801min, show D actinomycin D
Containing the homologue of actinomycin D in FGR, and according to the regression equation y=731.5x-17.709 of actinomycin D it is found that it contains
Amount is 0.015mg/mL；The present invention continues to have studied the dissolubility of D actinomycin D FGR and soda acid (pH), temperature, illumination (can
Light-exposed and ultraviolet light), metal ion, preservative, the physicochemical properties such as Oxidizing and Reducing Agents stability.
Detailed description of the invention
Fig. 1 is shown as the UV map of actinomycin D.
Fig. 2 is shown as the UV map of D actinomycin D FGR.
Fig. 3 is shown as the HPLC-UV detection of actinomycin D mark product.
Fig. 4 is shown as the HPLC-UV detection of D actinomycin D FGR.
Fig. 5 is shown as the efficient liquid phase separating spectrum of D actinomycin D FGR.
Fig. 6 is shown as influence of the pH to D actinomycin D FGR stability.
Fig. 7 is shown as influence (A: visible light of the light application time to D actinomycin D FGR stability；B: ultraviolet light).
Fig. 8 is shown as influence of the different temperatures to D actinomycin D FGR stability.
Fig. 9 is shown as influence of 100 DEG C of different times to D actinomycin D FGR stability.
Figure 10 is shown as influence of the metal ion to D actinomycin D FGR stability.
Figure 11 is shown as influence of the Oxidizing and Reducing Agents to D actinomycin D FGR stability.
Figure 12 is shown as influence of the preservative to D actinomycin D FGR stability.
Specific embodiment
A kind of method and its physicochemical property exploration of the initial gross separation identification of anti-cancer active matter D actinomycin D FGR, first
The anti-cancer active matter D actinomycin D FGR is swept into spectrum detection by ultraviolet specrophotometer (UV) all-wave length, finds absorption maximum
Wavelength is compared with object of suspicion actinomycin D, then further to actinomycin D and is put with high performance liquid chromatography (HPLC)
Line rhzomorph FGR is compared identification, then carries out initial gross separation to D actinomycin D FGR with high performance liquid chromatography (HPLC), most
The physicochemical property of the anti-cancer active matter D actinomycin D FGR is detected afterwards, dissolubility and acid including D actinomycin D FGR
Alkali (pH), temperature, illumination (visible light and ultraviolet light), metal ion, preservative, Oxidizing and Reducing Agents are to the shadow of its stability
It rings.
Embodiment 1:
The extraction of 1 D actinomycin D FGR
Actinomycetes fermentation liquor is taken out and shaken up, 4 DEG C of centrifugation 20min in the centrifuge of 4000r/min abandon precipitating, use
Supernatant is formulated as saturated common salt aqueous solution by salt, and using vacuum pump suction filtration, (saturated salt solution and vacuum filtration process can
Mitigate the emulsion of fermentation liquid), then supernatant and the ethyl acetate of 4 times of volumes are mixed well, 44 DEG C of water bath sonicators
30min is then moved into pears type separatory funnel, is stood 16h, is extracted with ethyl acetate, and organic phase is separated, until solution to be measured is
It is colourless, by organic phase reservation and -80 DEG C of freeze-drying 3d after 60 DEG C of decompression rotary evaporations, -20 DEG C freeze, to obtain thick
Extract D actinomycin D FGR is aubergine little particle.
The UV of 2 D actinomycin D FGR is detected
Actinomycin D is aubergine powder, there is certain viscosity, has antibacterial antitumaous effect, therefore suspect D actinomycin D FGR
For actinomycin D or its analog, therefore prepare actinomycin D mark product and carry out UV therewith to sweep spectrum comparison.Actinomycin D methanol is molten
Solution is the solution of 1mg/mL, and the D actinomycin D FGR for weighing 10mg is dissolved as the solution of 10mg/mL with methanol, does blank with methanol
Control carries out all-wave length (190~1000nm) on ultraviolet specrophotometer and sweeps spectrum, detects actinomycin D and actinomyces respectively
The maximum absorption wavelength of plain FGR.
Actinomycin D map such as Fig. 1, there are two actinomycin D maximum absorption wavelengths, respectively 252nm and 428nm.
D actinomycin D FGR map such as Fig. 2,190~1000nm of all-wave length sweep spectrum.The compound has maximum suction at 210nm
Receive wavelength, A210nm=3.42.
By Fig. 1 and 2 it is found that the same terms appearance time is inconsistent, it is seen that D actinomycin D FGR is not actinomycin D.
The HPLC of 3 D actinomycin D FGR is detected
Whether it is same substance further to verify D actinomycin D FGR and actinomycin D, therefore following HPLC is utilized to detect.
The preparation of 3.1 samples
D actinomycin D FGR is dissolved as the solution of 10mg/mL with methanol, spare after being filtered with 0.22 μm of filter；Actinomycin D
The solution of 1mg/mL is dissolved as with methanol, then successively doubling dilution is 0.5,0.25,0.125,0.0625,0.03125mg/mL
The solution of concentration, it is spare after being filtered respectively with 0.22 μm of filter.
The preparation of 3.2 mobile phases
Mobile phase: methanol (chromatographically pure) and fresh pure water (Wahaha) water use corresponding organic filter membrane and inorganic respectively
The vacuum pump of filter membrane filters 7~10 times, visually without visible foreign until, then ultrasound 30min is defoamed, and standing is spare to room temperature.
3.3 HPLC chromatogram testing conditions
Mobile phase is methanol: water=75:25, flow velocity 1mL/min, and sample volume is 10 μ L, and chromatographic column specification is Sunfire
C18 (250mm × 10mm), UV detector Detection wavelength are 252nm.
D actinomycin D FGR and actinomycin D elute 1h and 30min respectively, detect chromatogram, analysis D actinomycin D FGR and
The relationship of actinomycin D.
The HPLC testing result of 3.3 D actinomycin D FGR
Actinomycin D solution chromatogram such as Fig. 3 of 1mg/mL.Actinomycin D is in 10.589min appearance as shown in Figure 3.
D actinomycin D FGR is dissolved as the solution of 10mg/mL, HPLC detection with formic acid, DMSO and methanol respectively, and mobile phase is
Methanol: water=75:25, sample volume 10 μ L, Detection wavelength 252nm.As a result see Fig. 4.A, B, C respectively represent D actinomycin D in Fig. 4
The HPLC of the formic acid of FGR, DMSO and methanol solution detects figure: (A) formic acid and D actinomycin D FGR react, and solute effect is most
Good, no particle, solution is uniform, but peak position is in 2.025min out, inconsistent with actinomycin D；(B) DMSO solute effect
It, maximum peak appearance at 1.975min, but there is lesser peak to occur in 10~11min；(C) methanol solute effect is compared with first
Acid and DMSO take second place, and it is 2.577min and 3.506min that two maximums, which go out peak position, but as seen from the figure, at least 5 kinds or more
Peak, D actinomycin D FGR is cometabolism crude product, it at least contains 5 kinds or more substances.
4 D actinomycin D FGR initial gross separations
Initial gross separation is carried out to D actinomycin D FGR using HPLC eluent gradient elution method, gradient condition is shown in Table 1, other
HPLC testing conditions are constant, and concentration is the D actinomycin D FGR methanol solution of 10mg/mL, and sample volume is 10 μ L, and chromatographic column specification is
Sunfire C18 (250mm × 10mm), UV detector Detection wavelength are 252nm.30min, inspection are eluted according to the above chromatographic condition
Colour examining spectrogram.Table 1 is D actinomycin D FGR condition of gradient elution.
1 D actinomycin D FGR condition of gradient elution of table
| Time (min) | Methanol | Water | Flow velocity (mL/min) |
| 0 | 40 | 60 | 1 |
| 5 | 35 | 65 | 0.8 |
| 10 | 30 | 70 | 0.6 |
| 15 | 30 | 70 | 0.6 |
10 μ L of D actinomycin D FGR sample introduction, after gradient condition, testing result such as Fig. 5, totally 11 peaks in figure show unwrapping wire
At least 11 kinds or more of substance in this crude product of rhzomorph FGR, in combination with mass spectrum, infrared spectroscopy etc. to D actinomycin D FGR do into
Separates, purifies and identify to one step.
The dissolubility of 5 D actinomycin D FGR is analyzed
The D actinomycin D FGR of 10mg is soaked in a variety of 10mL organic solvent methanol, dehydrated alcohol, n-butanol, different respectively
Propyl alcohol, ethylene glycol, ethyl acetate, methylene chloride, chloroform ether, acetonitrile, normal propyl alcohol, 1- octanol, petroleum ether, benzene, acetic acid,
It in formic acid, ethyl alcohol, shakes up, observation dissolution situation after 6h.
It has been observed that there are apparent differences for the solubility being somebody's turn to do in the different organic reagent of polarity, as shown in table 2.It puts
Line rhzomorph FGR is insoluble in petroleum ether, ethyl acetate, chloroform, therefore D actinomycin D FGR is in the lesser solvent dissolution of polarity
It spends small；And solute effect is good in n-butanol, ethyl alcohol, the biggish solvent of methanol isopolarity, illustrates that D actinomycin D FGR is soluble in pole
The biggish organic solvent of property.Table 2 is the dissolution situation of D actinomycin D FGR.
The dissolution situation of 2 D actinomycin D FGR of table
| Solvent | Dissolve situation | Solvent | Dissolve situation |
| Methanol | Dissolution | Ether | Slightly soluble |
| Dehydrated alcohol | It is readily soluble | Acetonitrile | Slightly soluble |
| N-butanol | Dissolution | Normal propyl alcohol | Dissolution |
| Isopropanol | Dissolution | 1- octanol | Slightly soluble |
| Ethylene glycol | Dissolution | Petroleum ether | It is insoluble |
| Methylene chloride | It is insoluble | Benzene | It is readily soluble |
| Chloroform | It is insoluble | Acetic acid | Slightly soluble |
| Formic acid | It is readily soluble | Ethyl acetate | It is insoluble |
6 D actinomycin D FGR stability studies
Different temperatures, illumination, soda acid, Oxidizing and Reducing Agents, preservative and different metal ions are studied to actinomyces
The influence of plain FGR stability.
The ph stability of 6.1 D actinomycin D FGR
D actinomycin D FGR is formulated as to the solution of 0.1mg/mL with methanol, initial pH is 7.2, and average mark is loaded on 13 examinations
Its pH is adjusted to 2,3,4,5,6,7,8,9,10,11,12,13 and 14 respectively with HCl and NaOH solution, stands 3h, see by Guan Zhong
Examine solution colour variation.It is control with original D actinomycin D FGR, measures the absorbance value of maximum absorption wave strong point.
The original pH of D actinomycin D FGR is 7.2, as a result as shown in Figure 6: acid or alkali environment (5~12) of the pH near 7.2, right
It is all little in stability influence；It is under the conditions of peracid (pH < 5) instead, the substance is extremely unstable, and solution colour shoals；Cross alkali
Light absorption value is increased instead when (pH > 12), if D actinomycin D FGR is purely by way of a kind of haematochrome, effect is more preferable instead
A bit.
The photostability of 6.2 D actinomycin D FGR
6.2.1 influence of the visible light to D actinomycin D FGR stability
D actinomycin D FGR is formulated as to the solution of 0.1mg/mL with methanol, is equally divided into 15 parts, is placed in visible light according to growth
In case, carry out the visible lighting process of D actinomycin D FGR, the processing time is respectively 0,1, each processing of 2~15d set 3 repetitions,
To sample time, take out D actinomycin D FGR be stored under 4 DEG C of dark conditions, to gross sample after, with original actinomyces
Plain FGR is control, measures the absorption value of maximum absorption wave strong point.
6.2.2 influence of the ultraviolet light to D actinomycin D FGR stability
D actinomycin D FGR is formulated as to the solution of 0.1mg/mL with methanol, is equally divided into 5 parts, is put into 5 culture dishes,
Culture dish is exposed, and ultraviolet lamp directly shines, and is placed under ultraviolet lamp and irradiates 3,6,9,12 and for 24 hours respectively, is with original D actinomycin D FGR
Control measures the absorption value of maximum absorption wave strong point.
As a result see Fig. 7, it is seen that light does not have influence to D actinomycin D FGR substantially；Fig. 7 (B) is right in 0~9h of ultraviolet irradiation
D actinomycin D FGR stability there is not influence substantially, but absorption value reduces after > 9h, but influences yet little, about 0.03.
The thermal stability of 6.3 D actinomycin D FGR
6.3.1 the stability test of different heating temperature same time
D actinomycin D FGR is formulated as to the solution of 0.1mg/mL with methanol, is equally divided into 11 parts, with refrigerator, water-bath,
Autoclave adjusts 4,20,30,40,50,60,70,80,90,100 and 121 DEG C, takes out after each Temperature Treatment 30min, naturally cold
But, it is restored to initial volume, is control with original D actinomycin D FGR, measures the absorbance value of maximum absorption wave strong point.
As a result see Fig. 8, for temperature at 70 DEG C or less, temperature influences not significant (P > 0.05) to line rhzomorph FGR light absorption value,
When 70~120 DEG C, line rhzomorph FGR light absorption value is remarkably decreased (P < 0.05) as the temperature rises, and therefore, low temperature is to line bacterium
Plain FGR has substantially no effect on, and the ingredient of high temperature line rhzomorph FGR.
6.3.2 under the conditions of 100 DEG C the different disposal time stability test
D actinomycin D FGR is formulated as to the solution of 0.1mg/mL with methanol, is equally divided into 5 parts, in 100 DEG C of water-baths, in 15,
30, it is taken out after 60,90 and 120min, after cooling, add Reduction of methanol to initial volume, be control with original D actinomycin D FGR, survey
Measure the absorption value of maximum absorption wave strong point.
As a result as shown in figure 9, at 100 DEG C, the light absorption value of D actinomycin D FGR slowly reduces as the temperature rises and gradually
(P > 0.05) therefore although the ingredient of high temperature D actinomycin D FGR, continuous high temperature, will affect not significant.
Influence of 6.4 metal ions to D actinomycin D FGR stability
D actinomycin D FGR is formulated as the solution of 0.1mg/mL with methanol, is equally divided into 6 parts, is separately added into for 5 parts thereto
KCl、MnCl2、NaCl、NH4Cl、FeCl3、CuCl2、ZnCl2So that the concentration of metal ions of solution is 1mol/L, 12h is stood, with
It is remaining to measure the absorbance value of maximum absorption wave strong point with a for control, while measuring the anticancer effect of D actinomycin D FGR
Fruit.
The result is shown in Figure 10, influence D actinomycin D FGR light absorption value reduction metal ion by effect greatly to it is small successively are as follows: Mn2+
> K+> Fe3+；Promote the increased metal ion of D actinomycin D FGR light absorption value by effect greatly to it is small successively are as follows: Zn2+> Cu2+> Na+
> NH4 +.
The Oxidizing and Reducing Agents stability of 6.5 D actinomycin D FGR
D actinomycin D FGR is formulated as to the solution 60mL of 0.1mg/mL with methanol, pipettes 11 parts, every part of 5mL, chooses 10 parts
It is separately added into 20% nitric acid or concentrated hydrochloric acid solution 1,2,3,4,5m L, 11 parts are all supplied 10mL with methanol.2h is stood, with not
Add the D actinomycin D FGR solution of nitric acid or concentrated hydrochloric acid for control, measures the absorbance value of maximum absorption wave strong point.
As shown in Figure 11, reducing agent concentrated hydrochloric acid has an impact to the stability of D actinomycin D FGR；On the contrary, with oxidant (nitre
Acid) be continuously increased, the light absorption value of D actinomycin D FGR is bigger, shows that D actinomycin D FGR oxidation resistance is stronger.
Influence of 6.6 preservatives to D actinomycin D FGR stability
D actinomycin D FGR is formulated as to the solution of 0.1mg/mL with methanol, is equally divided into 13 parts, wherein 12 parts are separately added into
1,2, the salt of 4mg 0.5%, sucrose, brown sugar, sodium nitrite, stand 2h after mixing, and with remaining a for control, measurement is most
Absorption value at big absorbing wavelength.
As shown in Figure 12, the amount of addition and the addition of 4 kinds of preservatives (additive) all increases D actinomycin D FGR light absorption value
Greatly, therefore, if coming as pigment using adding these preservatives can be enhanced effect.Effect is descending successively are as follows: brown sugar >
Sodium nitrite > salt > sucrose.
Claims (6)
1. the isolation and identification method of anti-cancer active matter D actinomycin D FGR, which comprises the following steps:
1) D actinomycin D FGR is subjected to all-wave length by ultraviolet specrophotometer UV and sweeps spectrum detection；
2) D actinomycin D FGR is further identified with high-efficient liquid phase chromatogram HPLC again；
3) it is separated after identifying using high-efficient liquid phase chromatogram HPLC.
2. the isolation and identification method of anti-cancer active matter D actinomycin D FGR according to claim 1, which is characterized in that institute
The secondary metabolite that D actinomycin D FGR is bull streptomycete FGR-015 is stated, to expand medium liquid fermentation actinomyces FGR-
015 isolates and purifies gained；
The actinomyces are streptomycete, are named as bull streptomycete (Streptomyces tauricus) FGR-015, preservation list
Position is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the preservation time is on June 14th, 2018, and preservation is compiled
Number be CGMCC No.15942；
The D actinomycin D FGR is bull actinomyces FGR-015 secondary metabolite, to expand medium liquid fermentation actinomyces
FGR-015 isolates and purifies gained；
The composition for expanding culture medium are as follows: soluble starch 20g, KNO3 0.1g, K2HPO40.05g, MgSO4·7H2O
0.05g, NaCl 0.05g, FeSO4·7H2O 0.001g, distilled water 1000mL；PH value is adjusted to 7.1；
The expansion fermentation process are as follows: inoculum concentration 12.61%, bottling amount are 48.65%, and shaking speed 150r/min shakes
Bed tempertaure is 28 DEG C, fermentation time 7.5d；
The isolation and purification method, comprising the following steps: centrifugal rotational speed 10000r/min, 4 DEG C of centrifugation 20min abandon sediment,
It is extracted by extractant of ethyl acetate, 30 DEG C of water bath sonicator 30min are then moved into pears type separatory funnel, stand 2h；With acetic acid
Ethyl ester is extractant, and solid-liquid ratio 1:5, ultrasonic temperature is 44 DEG C, extraction time 16h；It is extracted with ethyl acetate 3 times；Separation
Organic phase, until solution to be measured be it is colourless, organic phase is retained and pass through 60 DEG C of rotary evaporations, -20 DEG C icings and -80 DEG C freeze
D actinomycin D FGR finished product is made in dry 3d.
3. the isolation and identification method of anti-cancer active matter D actinomycin D FGR according to claim 2, which is characterized in that institute
It states in step 1), it is maximum absorption wavelength 210nm that D actinomycin D FGR ultraviolet specrophotometer, which sweeps spectrum result,.
4. the isolation and identification method of anti-cancer active matter D actinomycin D FGR according to claim 3, which is characterized in that institute
It states in step 1), D actinomycin D FGR ultraviolet specrophotometer sweeps spectrum method particularly includes: weigh 10mg D actinomycin D FGR first
Alcohol is dissolved as the solution of 10mg/mL, does blank control with methanol, and all-wave length is carried out on ultraviolet specrophotometer and sweeps spectrum, i.e., 190
~1000nm sweeps spectrum, detects the maximum absorption wavelength of D actinomycin D FGR.
5. the isolation and identification method of anti-cancer active matter D actinomycin D FGR according to claim 4, which is characterized in that institute
It states in step 2), the high-efficient liquid phase chromatogram HPLC qualification result of D actinomycin D FGR is the substance containing 11 kinds or more.
6. the isolation and identification method of anti-cancer active matter D actinomycin D FGR according to claim 5, which is characterized in that institute
It states in step 2), the concrete operations of high performance liquid chromatography are as follows: the D actinomycin D FGR of 10mg is dissolved as 10mg/mL's with methanol
Solution；The mobile phase methanol and pure water of chromatographically pure are then ultrasonic respectively with vacuum pump filtering 7~10 times to complete free from admixture
30min, rest chamber warm standby are used；HPLC testing conditions are as follows: mobile phase is methanol: water=75:25, flow velocity 1mL/min elute 1h,
Sample volume is 10 μ L, and chromatographic column specification is PursuitXRs C18 (250mm × 10mm), and UV detector Detection wavelength is 252nm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656131A (en) * | 2019-03-05 | 2020-01-07 | 西藏农牧学院 | Preparation method of antibacterial secondary metabolite of actinomycetes |
| CN112010942A (en) * | 2020-08-28 | 2020-12-01 | 深圳大学 | Method for preparing actinomycin D and actinomycin X by using supercritical fluid chromatography2Method of producing a composite material |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104995518A (en) * | 2012-07-24 | 2015-10-21 | 米纳瓦生物技术公司 | NME variant species expression and suppression |
| US10036048B1 (en) * | 2017-09-28 | 2018-07-31 | King Saud University | Process for obtaining a naphthoquinone derivative from Streptomyces sp |
-
2018
- 2018-08-01 CN CN201810866167.4A patent/CN109081863A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104995518A (en) * | 2012-07-24 | 2015-10-21 | 米纳瓦生物技术公司 | NME variant species expression and suppression |
| US10036048B1 (en) * | 2017-09-28 | 2018-07-31 | King Saud University | Process for obtaining a naphthoquinone derivative from Streptomyces sp |
Non-Patent Citations (2)
| Title |
|---|
| SHABIR AHMAD RATHER 等: "Isolation and characterization of Streptomyces tauricus from Thajiwas glacier—a new source of actinomycin-D", 《MEDICINAL CHEMISTRY RESEARCH》 * |
| 曾伟: "海洋放线菌抗肿瘤活性菌株的筛选及菌株N350抗肿瘤活性物质的研究", 《中国优秀博硕士学位论文全文数据库（博士） 医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110656131A (en) * | 2019-03-05 | 2020-01-07 | 西藏农牧学院 | Preparation method of antibacterial secondary metabolite of actinomycetes |
| CN112010942A (en) * | 2020-08-28 | 2020-12-01 | 深圳大学 | Method for preparing actinomycin D and actinomycin X by using supercritical fluid chromatography2Method of producing a composite material |
| CN112010942B (en) * | 2020-08-28 | 2022-05-17 | 深圳大学 | Method for preparing actinomycin D and actinomycin X by using supercritical fluid chromatography2Method |
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Application publication date: 20181225 |