CN105061443B - Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting human liver cancer - Google Patents
Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting human liver cancer Download PDFInfo
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- CN105061443B CN105061443B CN201510422116.9A CN201510422116A CN105061443B CN 105061443 B CN105061443 B CN 105061443B CN 201510422116 A CN201510422116 A CN 201510422116A CN 105061443 B CN105061443 B CN 105061443B
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- liver cancer
- human liver
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- penicitrinine
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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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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention relates to penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting human liver cancer. The compound has the effect of restraining human liver cancer cell proliferation. The structural formula of the compound is shown in the description. According to the invention, through fermenting culture of penicillium citrinum IBPT-5, a fermentation product is obtained, and then the compound is separated and purified from the fermentation product. Experiment results confirm that the compound has better antitumor activity on human liver cancer cells such as PLC/PRF/5, HUH-7 and SK-HEP-1. The penicillium citrinum-derived penicitrinine A can be applied to preparation of human liver cancer cell proliferation inhibition drugs or antitumor drugs to be used for research on human liver cancer.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of penicitrinine A for coming from Aspergillus citrimum and its preparation are anti-
The application of human liver 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 and there is a title of plant alkaloid.Alkaloid has important physiological action to humans and animals, including flat
Antitussive, blood sugar lowering, blood fat reducing, antibacterial, antitumor, analgesia etc. are breathed heavily, wherein projecting the most with antibacterial, anti-tumor activity.Natural knot
Structure alkaloid is the important sources that lead compound is found in innovation drug research, and the alkaloidal drug of clinic has been applied at present
Nearly hundred kinds of Jing.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, Aspergillus citrimum (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) crude extract of tunning has good cell inhibitory effect activity, and its active component is studied then.
Research finds that shown alkaloid compound has anti-human liver cancer activity, has not yet to see the compound to human liver cancer cell PLC/
The report of the proliferation inhibition activity of PRF/5, HUH-7, SK-HEP-1, therefore also there is not yet medicine related to this on market.
The content of the invention
It is an object of the invention to provide a kind of penicitrinine A for coming from Aspergillus citrimum and its preparing anti-human liver cancer medicine
The application of thing.The compound has suppression human hepatoma cell proliferation effect, with anti-human liver cancer activity.Its structural formula is：
。
The preparation method of the compound, is by fermentation culture Aspergillus citrimum (Penicillium citrinum) IBPT-
5, fermented product is obtained, the compound is then isolated and purified out from fermented product.Comprise the following steps that：
1 fermenting and producing
The conventional method of cultivating microorganism, takes Aspergillus citrimum (Penicillium citrinum) IBPT-5 and is inoculated into PDA
Cultivate 4 days in 28 DEG C of incubators on solid slant culture base, in being then seeded into culture fluid, 28 DEG C of static gas wave refrigerators are after 30 days,
Obtain mycelium and fermentation liquid；The culture fluid 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 extractum
With gauze by mycelium and separation of fermentative broth.By fermentation liquid ethyl acetate 1:2 (v/v) are extracted twice, extraction
Liquid vacuum distillation obtains the g of ethyl acetate extract 32.0 of fermentation liquid to dry.
The separation and purification of 3 compounds
The extractum after 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol is eluent decompression silicon
Glue chromatographic column gradient elution, obtains 11 components.Component 7 (4.2 g) (dichloromethane：Methanol v/v=100:1 eluate)
With dichloromethane：Methanol is eluent, further by pressurized silica gel column chromatography gradient elution, the subfraction 7-6 (two for obtaining
Chloromethanes：Methanol v/v=50:1 eluate) by semi-preparative liquid chromatography (1010 types 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)。
Aspergillus citrimum (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.
Described compound prepare suppress human hepatoma cell proliferation medicine in purposes, and the compound prepare it is anti-human
Purposes in liver-cancer medicine.The tumor cell is human liver cancer cell PLC/PRF/5, HUH-7, SK-HEP-1.
The remarkable advantage of the present invention：The alkaloid compound shown in research is more rare, the alkaloid compound tool
Have it is significant suppress human hepatoma cell proliferation activity, have not yet to see the compound to human liver cancer cell PLC/PRF/5, HUH-7,
The report of SK-HEP-1 proliferation inhibition activities, therefore also there is not yet medicine related to this on market.
Description of the drawings
Fig. 1 Penicitrinine A main COSY, HMBC and NOESY signal.
Specific embodiment
The chemical constitution of the compound of indication in examples below：
。
The fermenting and producing and separation and purification of embodiment 1 compound
1 fermenting and producing
The fermentation culture of production bacterium：By the conventional method of cultivating microorganism, Aspergillus citrimum 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 on PDA solid slant culture bases in 28 DEG C of incubators
Culture 4 days.
Take the slant culture Aspergillus citrimum of 4 days（Penicillium citrinum）IBPT-5 is inoculated into equipped with 400mL cultures
[culture fluid 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 fermentation liquid.
The acquisition of 2 extractum
With gauze by mycelium and separation of fermentative broth.By fermentation liquid ethyl acetate 1:2 (v/v) are extracted twice, extraction
Liquid vacuum distillation obtains the g of ethyl acetate extract 32.0 of fermentation liquid to dry.
The separation and purification of 3 compounds
The extractum after 200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol is eluent decompression silica gel color
Spectrum post gradient elution, obtains 11 components.Component 7 (4.2 g) (dichloromethane：Methanol v/v=100:1 eluate) with two
Chloromethanes：Methanol is eluent, further by pressurized silica gel column chromatography gradient elution, the subfraction 7-6 (dichloromethanes for obtaining
Alkane：Methanol v/v=50:1 eluate) by semi-preparative liquid chromatography (1010 types 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 be given at m/z 484.2711 molecular ion peak [M-
H]–, (calcd. for C28H38NO6, 484.2705), point out molecular weight to be 485, speculate 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 enforcement 1.Precision is weighed
Appropriate amount of sample, with DMSO the solution of desired concn is configured to, for surveying activity.
The successive transfer culture of cell line and cell adopts tumor cell line, DMEM culture medium of the tumor cell containing 10% FBS,
At 37 DEG C in being passed through 5% CO2Incubator in successive transfer culture.
Cell inhibitory effect activity test method
WST-1 methods are taken the logarithm the tumor cell of trophophase, and cell density is adjusted to into per milliliter 5 × 104Individual cell, by every
The μ L of hole 100 are inoculated in 96 porocyte culture plates, and in 37 DEG C 5% CO is passed through2Incubator 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 microplate reader and determine per light absorption value (OD) value of the 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, according to the Cytostatic to tumor cell rate of the compound of variable 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 people's human hepatoma cell proliferation
3. conclusion
The compound has preferable anti-tumor activity to human liver cancer cell PLC/PRF/5, HUH-7, SK-HEP-1.Can make
Medicine or antitumor drug is suppressed to be used for the research of human liver cancer to prepare human hepatoma cell proliferation.
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 the covering scope of the present invention.
Claims (3)
1. the penicitrinine A of Aspergillus citrimum are come fromSuppress people preparing
Application in hepatoma cell proliferation medicine.
2. application according to claim 1, it is characterised in that：Described human liver cancer cell is human liver cancer cell PLC/PRF/
5、HUH-7、SK-HEP-1。
3. compoundApplication in anti-human liver cancer medicine is prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510422116.9A CN105061443B (en) | 2015-07-17 | 2015-07-17 | Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting human liver cancer |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510422116.9A CN105061443B (en) | 2015-07-17 | 2015-07-17 | Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting human liver cancer |
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| CN105061443A CN105061443A (en) | 2015-11-18 |
| CN105061443B true CN105061443B (en) | 2017-04-26 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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