CN105153176B - The penicitrinine A for coming from Penicillium citrinum and its application for preparing anti-human lung-cancer medicament - Google Patents
The penicitrinine A for coming from Penicillium citrinum and its application for preparing anti-human lung-cancer medicament Download PDFInfo
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- CN105153176B CN105153176B CN201510423833.3A CN201510423833A CN105153176B CN 105153176 B CN105153176 B CN 105153176B CN 201510423833 A CN201510423833 A CN 201510423833A CN 105153176 B CN105153176 B CN 105153176B
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- human lung
- penicillium citrinum
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- penicitrinine
- carcinoma cell
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
- C07D491/20—Spiro-condensed systems
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
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Abstract
The present invention relates to a kind of penicitrinine A for coming from Penicillium citrinum and its application for preparing anti-human lung-cancer medicament.The compound has suppression human lung carcinoma cell proliferation function.Its structural formula is：.By fermented and cultured Penicillium citrinum (Penicillium citrinum) IBPT 5, fermentate is obtained, the compound is then isolated and purified out from fermentate.It is verified by experiments, the compound has preferable antitumor activity to human lung carcinoma cell 95 D, SK MES 1, SPC A1, A 549.The research of human lung cancer can be used for as human lung carcinoma cell Proliferation Ability medicine or antineoplastic is prepared.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of penicitrinine A for coming from Penicillium citrinum and its preparation are anti-
The application of human lung cancer medicine.
Background technology
Alkaloid is the organic compounds containing nitrogen that a class is produced by biological cometabolism, the alkaloid species in nature compared with
It is many, mostly from plant, therefore there is the title of vegetable soda again.Alkaloid has important physiological action to humans and animals, including flat
Antibechic, hypoglycemic, reducing blood lipid, antibacterial, antitumor, analgesia etc. are breathed heavily, wherein being protruded the most with antibacterial, antitumor activity.Natural knot
Structure alkaloid is the important sources of discovery lead compound in innovation drug research, and the alkaloidal drug of clinic has been applied at present
Through nearly hundred kinds.Research finds that some marine fungis can produce the biology that structure is novel, activity is good during cometabolism
Alkali, with good medicinal and industrialization prospect.
The present inventor studies and learns, Penicillium citrinum (Penicillium citrinum) IBPT-5, (in 2013 12
The moon is deposited in China typical culture collection center, address on 25th:Wuhan Wuhan University, deposit number is：CCTCC NO：M
2013713) CE of tunning has good cell inhibitory effect activity, and its active component is studied then.
Research finds that shown alkaloid compound has anti-human lung cancer activity, has not yet to see the compound to human lung carcinoma cell 95-
The report of the proliferation inhibition activity of D, SK-MES-1, SPC-A1, A-549, therefore in the market is also there is not yet medicine related to this
Thing.
The content of the invention
It is an object of the invention to provide a kind of penicitrinine A for coming from Penicillium citrinum and its prepare anti-human lung cancer drug
The application of thing.The compound has suppression human lung carcinoma cell proliferation function, with anti-human lung cancer activity.Its structural formula is：
。
The preparation method of the compound, be by fermented and cultured Penicillium citrinum (Penicillium citrinum) IBPT-
5, fermentate is obtained, the compound is then isolated and purified out from fermentate.Comprise the following steps that：
1 fermenting and producing
Cultivate microorganism conventional method, take Penicillium citrinum (Penicillium citrinum) IBPT-5 is inoculated into PDA
Cultivated 4 days in 28 DEG C of incubators in solid slope culture medium, be then seeded into nutrient solution, 28 DEG C of static gas wave refrigerators are after 30 days,
Obtain mycelium and zymotic fluid；The nutrient solution composition：Every liter of water contains the g of mannitol 20.0, the g of yeast extract 3.0, maltose 20.0
G, the g of monosodium glutamate 10.0, glucose 10.0 g, KH2PO4 0.5 g、MgSO40.3 g、NaCl 30.0 g；
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.By zymotic fluid ethyl acetate 1:2 (v/v) are extracted twice, extraction
Liquid vacuum distillation obtains the g of ethyl acetate extract 32.0 of zymotic fluid to dry.
The separation and purification of 3 compounds
The medicinal extract by after 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methyl alcohol is eluent decompression silicon
Glue chromatographic column gradient elution, obtains 11 components.Component 7 (4.2 g) (dichloromethane：Methyl alcohol v/v=100:1 eluate)
With dichloromethane：Methyl alcohol is eluent, further by pressurized silica gel column chromatography gradient elution, the subfraction 7-6 (two for obtaining
Chloromethanes：Methyl alcohol v/v=50:1 eluate) by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm)：
Separation flow velocity is 5 mL/min, and mobile phase is that 75% acetonitrile contains 0.1% TFA, obtains shown compound (97.9 mg, tR 19.3
min)。
Penicillium citrinum (Penicillium citrinum) IBPT-5, is deposited in China on December 25th, 2013
Type Tissue Collection, address:Wuhan Wuhan University, deposit number is：CCTCC NO：M 2013713.
The present invention also protects described compound preparing the purposes in suppressing human lung carcinoma cell hyperproliferation agent, and the change
Purposes of the compound in anti-human lung-cancer medicament is prepared.The tumour cell be human lung carcinoma cell 95-D, SK-MES-1, SPC-A1,
A-549。
Remarkable advantage of the invention：The alkaloid compound shown in research is more rare, the alkaloid compound tool
Have it is significant suppress human lung carcinoma cell proliferation activity, have not yet to see the compound to human lung carcinoma cell 95-D, SK-MES-1,
The report of SPC-A1, A-549 proliferation inhibition activity, therefore in the market is also there is not yet medicine related to this.
Brief description of the drawings
Fig. 1 Penicitrinine A main COSY, HMBC and NOESY signal.
Specific embodiment
The chemical constitution of signified compound in examples below：
。
The fermenting and producing and separation and purification of embodiment 1 compound
1 fermenting and producing
Produce the fermented and cultured of bacterium：By the conventional method of culture microorganism, Penicillium citrinum is taken（Penicillium citrinum）IBPT-5 (is deposited in China typical culture collection center, address on December 25th, 2013:Wuhan is military
Chinese university, deposit number is：CCTCC NO：M 2013713) it is inoculated into PDA solid slope culture mediums in 28 DEG C of incubators
Culture 4 days.
Take the inclined-plane culture Penicillium citrinum of 4 days（Penicillium citrinum）IBPT-5 is inoculated into equipped with 400mL cultures
[nutrient solution constitutes (g/l) to liquid：Mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0,
KH2PO40.5, MgSO40.3, NaCl 30.0 constant volume] 1000mL conical flasks in, 28 DEG C of static gas wave refrigerators are after 30 days, obtain
Mycelium and zymotic fluid.
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.By zymotic fluid ethyl acetate 1:2 (v/v) are extracted twice, extraction
Liquid vacuum distillation obtains the g of ethyl acetate extract 32.0 of zymotic fluid to dry.
The separation and purification of 3 compounds
The medicinal extract by after 200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methyl alcohol is eluent decompression silica gel color
Spectrum post gradient elution, obtains 11 components.Component 7 (4.2 g) (dichloromethane：Methyl alcohol v/v=100:1 eluate) with two
Chloromethanes：Methyl alcohol is eluent, further by pressurized silica gel column chromatography gradient elution, the subfraction 7-6 (dichloromethanes for obtaining
Alkane：Methyl alcohol v/v=50:1 eluate) by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm)：Separate
Flow velocity is 5 mL/min, and mobile phase is that 75% acetonitrile contains 0.1% TFA, obtains shown compound (97.9 mg, tR 19.3
min)。
Compound as yellow oily, high resolution mass spectrum HRESI-MS existsm/zBe given at 484.2711 molecular ion peak [M-
H]–, (calcd. for C28H38NO6, 484.2705), it is 485 to point out molecular weight, speculates that molecular formula is with reference to spectral information
C28H39NO6。1H and13C-NMR data are shown in Table 1, and main COSY, HMBC and NOESY signals are shown in Fig. 1.
The NMR compounds of table 11H and13C-NMR data (500MHz1H and 125MHz 13C, in CDCl3)
The test of the anti tumor activity in vitro of embodiment 2
1 laboratory sample and experimental technique
The preparation test sample of sample solution is the pure compounds of separation and purification in above-mentioned implementation 1.Precision is weighed
Appropriate amount of sample, the solution of required concentration is configured to DMSO, for surveying activity.
The squamous subculture of cell line and cell uses tumor cell line, tumour cell to be trained with the RPMI 1640 containing 10% FBS
Base is supported, at 37 DEG C in being passed through 5% CO2Incubator in squamous subculture.
Cell inhibitory effect activity test method
WST-1 methods are taken the logarithm the tumour cell in growth period, and cell density is adjusted into every milliliter 5 × 104Individual cell, by every
The μ L of hole 100 are inoculated in 96 porocyte culture plates, and 5% CO is passed through in 37 DEG C2Incubator in overnight incubation.Suck supernatant,
The μ L of culture medium 100 containing sample are added, continues to cultivate 48 h.10 μ L WST-1 liquid are added per hole, 4 h are cultivated.Using Bio-
Rad companies produce 680 type ELIASAs and determine per light absorption value (OD) value of hole at 450nm.Sample is every in the orifice plate of same 96
Individual concentration is respectively provided with five holes, separately sets five hole blanks and acellular zeroing hole (if to have color to do relative medicine dense for medicine
Spend acellular zeroing).Each hole OD values first do corresponding acellular zeroing, then take five hole mean OD values by IR (%)=(ODBlank-
ODSample)/ODBlank× 100% formula calculates cell proliferation inhibition rate (IR%) under each concentration.
2. experimental result
Cell inhibitory effect active testing result
In the test of WST-1 methods, the Cytostatic to tumor cell rate of the compound according to various concentrations, application
SPSS16.0 softwares carry out data processing and calculation of half inhibitory concentration IC50Value.The results are shown in Table 2.
Inhibitory activity of the compound of table 2 to everybody proliferation of lung cancer cells
3. conclusion
The compound has preferable antitumor activity to human lung carcinoma cell 95-D, SK-MES-1, SPC-A1, A-549.Can
It is used for the research of human lung cancer as human lung carcinoma cell Proliferation Ability medicine or antineoplastic is prepared.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to covering scope of the invention.
Claims (3)
1. the penicitrinine A of Penicillium citrinum are come fromSuppress people preparing
Application in proliferation of lung cancer cells medicine.
2. application according to claim 1, it is characterised in that：Described human lung carcinoma cell is human lung carcinoma cell 95-D, SK-
MES-1、SPC-A1、A-549。
3. compoundApplication in anti-human lung-cancer medicament is prepared.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510423833.3A CN105153176B (en) | 2015-07-17 | 2015-07-17 | The penicitrinine A for coming from Penicillium citrinum and its application for preparing anti-human lung-cancer medicament |
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| CN201510423833.3A CN105153176B (en) | 2015-07-17 | 2015-07-17 | The penicitrinine A for coming from Penicillium citrinum and its application for preparing anti-human lung-cancer medicament |
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| CN105153176A CN105153176A (en) | 2015-12-16 |
| CN105153176B true CN105153176B (en) | 2017-06-06 |
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| ES2274236T3 (en) * | 2002-06-20 | 2007-05-16 | Astion Dermatology A/S | NOVEDOUS COMPLEXES OF ESTERES OF FATTY ACIDS OF POLYHYDROXIALCANS AND NACINAMIDE. |
| WO2006075395A1 (en) * | 2005-01-11 | 2006-07-20 | The Kitasato Institute | β-LACTAM ANTIBIOTIC ACTIVITY ENHANCER AND PROCESS FOR PRODUCING THE SAME |
| CN104478891B (en) * | 2014-12-18 | 2016-08-24 | 福州大学 | Citrinin compounds penicitrinol O coming from Aspergillus citrimum and its preparation method and application |
| CN105061446B (en) * | 2015-07-17 | 2017-04-26 | 福建省肿瘤医院 | Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting nasopharyngeal carcinoma |
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