CN107325087B - Citrinin compounds dicitrinone D and its application in terms of malignant mela noma - Google Patents
Citrinin compounds dicitrinone D and its application in terms of malignant mela noma Download PDFInfo
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- CN107325087B CN107325087B CN201610361240.3A CN201610361240A CN107325087B CN 107325087 B CN107325087 B CN 107325087B CN 201610361240 A CN201610361240 A CN 201610361240A CN 107325087 B CN107325087 B CN 107325087B
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- dicitrinone
- citrinin
- citrinin compounds
- penicillium citrinum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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- 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
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
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Abstract
The invention discloses a kind of citrinin compounds dicitrinone D and its application in terms of malignant mela noma, structural formulas are as follows:.By fermented and cultured Penicillium citrinum (Penicillium citrinum) IBPT-5, fermentation material is obtained, the compound is then isolated and purified out from fermentation material.It is verified by experiments, which has preferable anti-tumor activity to people's Human melanoma cell line A375, can be used as the research for preparing malignant melanoma cell Proliferation Ability drug or anti-tumor drug for malignant mela noma.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of citrinin compounds dicitrinone D and its
Application in terms of malignant mela noma.
Background technique
Citrinin is a kind of organic compounds containing nitrogen generated by biological cometabolism, the citrinin type in nature compared with
It is more, greatly mostly from plant, therefore there is the title of vegetable soda again.Citrinin has important physiological action to humans and animals, including flat
Antibechic, hypoglycemic, reducing blood lipid, antibacterial, antitumor, analgesia etc. are breathed heavily, wherein the most prominent with antibacterial, anti-tumor activity.Natural knot
Structure citrinin is the important sources that lead compound is found in innovation drug research, has been applied to clinical citrinin drug at present
Through nearly hundred kinds.The study found that can to generate structure novel, activity good tangerine during cometabolism mould for some marine fungis
Element has 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 on 25th, and address: Wuhan Wuhan University, deposit number are: CCTCC NO:M
2013713) crude extract of tunning has good cell inhibitory effect activity, studies then its active constituent.
Research finds that shown citrinin compounds have anti-malignant mela noma activity, and it is pernicious to people black to have not yet to see the compound
The report of the proliferation inhibition activity of pigment oncocyte A375, therefore in the market also there is not yet drug related to this.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation sides citrinin compounds dicitrinone D derived from Penicillium citrinum
Method and its application for inhibiting malignant melanoma cell proliferation aspect, which, which has, inhibits malignant melanoma cell proliferation
Effect has anti-malignant mela noma activity, structural formula are as follows:
。
The preparation method of the compound, be by fermented and cultured Penicillium citrinum (Penicillium citrinum) IBPT-
5, fermentation material is obtained, the compound is then isolated and purified out from fermentation material, the specific steps are as follows:
(1) fermenting and producing
Cultivate microorganism conventional method, take Penicillium citrinum (Penicillium citrinum) IBPT-5 is inoculated into PDA
It cultivates 4 days, is then seeded into culture solution in 28 DEG C of incubators in solid slope culture medium, 28 DEG C after static gas wave refrigerator 30 days,
Obtain mycelium and fermentation liquid；The culture solution composition: every liter of water contains 20.0 g of mannitol, 3.0 g of yeast extract, maltose 20.0
G, 10.0 g of monosodium glutamate, glucose 10.0 g, KH2PO4 0.5 g、MgSO430.0 g of 0.3 g and NaCl；
(2) acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth, the ethyl acetate of fermentation liquid two volumes is extracted twice, is extracted
Liquid is evaporated under reduced pressure to doing, and obtains the ethyl acetate extract of fermentation liquid；
(3) separation and purification of compound
After the ethyl acetate extract that the step (2) obtains passes through 100-200 mesh silica gel mixed sample, with petroleum ether: methylene chloride:
Methanol is that eluent depressurizes silica gel chromatographic column gradient elution, using methylene chloride: methanol further passes through pressurization silicon as eluent
Plastic column chromatography gradient elution, obtains methylene chloride: the eluate of methanol v/v=100:1, then passes through 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 85% acetonitrile contains 0.1% TFA, obtain shown in
Compound, tR 6.8 min。
Penicillium citrinum (Penicillium citrinum) IBPT-5, is deposited in China on December 25th, 2013
Type Tissue Collection, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013713.
The present invention also protects the compound to inhibit the purposes in malignant melanoma cell hyperproliferation agent in preparation,
And the compound is that people's malignant mela noma is thin preparing the purposes in anti-malignant mela noma drug, the melanoma cells
Born of the same parents A375.
The beneficial effects of the present invention are: the citrinin compound shown in studying is more rare, the citrinin class chemical combination
Object has significant inhibition malignant melanoma cell proliferation activity, and it is thin to people's malignant mela noma to have not yet to see the compound
The report of born of the same parents' A375 proliferation inhibition activity, therefore in the market also there is not yet drug related to this.
Detailed description of the invention
Fig. 1 is the main COSY of Dicitrinone D, HMBC and NOE signal.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not limited only to these embodiments.
The chemical structure of signified compound in the following example:
。
The fermenting and producing and separation and purification of 1 compound of embodiment
1 fermenting and producing
Produce bacterium fermented and cultured: by culture microorganism conventional method, take Penicillium citrinum (Penicillium citrinum) (be deposited in China typical culture collection center on December 25th, 2013, address: Wuhan is military by IBPT-5
Chinese university, deposit number are: CCTCC NO:M 2013713) in right amount, it is inoculated into PDA solid slope culture medium and is trained at 28 DEG C
It supports and is cultivated 4 days in case.
Take 4 days Penicillium citrinums of inclined-plane culture (Penicillium citrinum) appropriate IBPT-5, it is inoculated into equipped with 400mL
[culture solution forms (grams per liter) to culture solution: 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 flask in, 28 DEG C after static gas wave refrigerator 30 days,
Obtain mycelium and fermentation liquid.
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.Fermentation liquid is extracted twice with ethyl acetate 1:2 (v/v), is extracted
Liquid is evaporated under reduced pressure to doing, and obtains 32.0 g of ethyl acetate extract of fermentation liquid.
The separation and purification of 3 compounds
After the medicinal extract passes through 100-200 mesh silica gel mixed sample, using petroleum ether: methylene chloride: methanol depressurizes silicon as eluent
Glue chromatographic column gradient elution obtains 11 components.Component 3 (1.8 g) (eluate of methylene chloride) is with methylene chloride: methanol
For eluent, further eluted by pressurized silica gel column chromatographic grade, obtained subfraction 3-2 (210 mg) (methylene chloride:
The eluate of methanol v/v=100:1), then pass through 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 85% acetonitrile contains 0.1% TFA, obtains shown compound (4.6 mg, tR 6.8 min)。
Compound as white powder, high resolution mass spectrum HRESI-MS existm/z359.1457 place provide molecular ion peak [M+
H]+, (calcd. for C20H23O6, 359.1489), prompting molecular weight is 358, speculates that molecular formula is in conjunction with spectral information
C20H22O6。1H and13C-NMR data are shown in Table 1, and main COSY, HMBC and NOE signal is shown in Fig. 1.
1 NMR compound of table1H and13C-NMR data (500MHz1H and 125MHz 13C, in CDCl3)
The test of 2 anti tumor activity in vitro of embodiment
1 laboratory sample and experimental method
The preparation test sample of sample solution is the pure compounds of separation and purification in above-mentioned implementation 1.Precision weighs
Appropriate amount of sample is configured to the solution of required concentration with DMSO, for surveying activity.
The squamous subculture of cell line and cell uses tumor cell line, and malignant melanoma cell is used containing 10% FBS
1640 culture medium of RPMI, at 37 DEG C in being passed through 5% CO2Incubator in squamous subculture.
Cell inhibitory effect activity test method
Cell density is adjusted to every milliliter 5 × 10 by the tumour cell of WST-1 method logarithmic growth phase4A cell, by every
100 μ L of hole is inoculated in 96 porocyte culture plates, is passed through 5% CO in 37 DEG C2Incubator in overnight incubation.Supernatant is sucked,
100 μ L of culture medium with sample is added, continues to cultivate 48 h.10 μ L WST-1 liquid are added in every hole, cultivate 4 h.Utilize Bio-
Rad company produces 680 type microplate reader and measures light absorption value (OD) value of every hole at 450nm.Sample is every in 96 orifice plate of same
A concentration is respectively provided with five holes, separately sets five hole blank controls and cell-free zeroing hole (if to have color to do relative medicine dense for drug
Spend cell-free zeroing).Each hole OD value first does corresponding cell-free zeroing, then takes five hole mean OD values by IR (%)=(ODBlank control-
ODSample)/ODBlank control× 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 method, according to the malignant melanoma cell proliferation inhibition rate of the compound of various concentration, answer
Data processing and calculation of half inhibitory concentration IC are carried out with SPSS16.0 software50Value.It the results are shown in Table 2.
The inhibitory activity that 2 compound of table is proliferated people's malignant melanoma cell
3. conclusion
The compound has preferable anti-tumor activity to people's Human melanoma cell line A375.Can be used as prepare it is pernicious black
Melanoma cytostatic object or anti-tumor drug are used for the research of malignant mela noma.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of application of citrinin compounds dicitrinone D, it is characterised in that: the citrinin compounds
The structural formula of dicitrinone D is, the citrinin compounds dicitrinone
D is applied to preparation and inhibits malignant melanoma cell hyperproliferation agent；The citrinin compounds dicitrinone D is to pass through
Fermented and cultured Penicillium citrinum (Penicillium citrinum) IBPT-5, fermentation material is obtained, is then separated from fermentation material pure
Dissolve the compound.
2. a kind of application of citrinin compounds dicitrinone D, it is characterised in that: the citrinin compounds
The structural formula of dicitrinone D is, the citrinin compounds dicitrinone
D is applied to prepare anti-malignant mela noma drug；The citrinin compounds dicitrinone D is by fermented and cultured tangerine
Mould (Penicillium citrinum) IBPT-5, fermentation material is obtained, the chemical combination is then isolated and purified out from fermentation material
Object.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104402898A (en) * | 2014-12-18 | 2015-03-11 | 福州大学 | Citrinin compound penicitrinol M derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol M |
| CN104402899A (en) * | 2014-12-18 | 2015-03-11 | 福州大学 | Citrinin compound penicitrinol L derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol L |
| CN104447781A (en) * | 2014-12-18 | 2015-03-25 | 福州大学 | Citrinin compound penicitrinol N derived from penicillium citrinum as well as preparation method and application thereof |
| CN104478891A (en) * | 2014-12-18 | 2015-04-01 | 福州大学 | Penicillium citrinum sourced citrinin compound (penicitrinol O) as well as preparation method and application thereof |
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- 2016-05-27 CN CN201610361240.3A patent/CN107325087B/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104402898A (en) * | 2014-12-18 | 2015-03-11 | 福州大学 | Citrinin compound penicitrinol M derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol M |
| CN104402899A (en) * | 2014-12-18 | 2015-03-11 | 福州大学 | Citrinin compound penicitrinol L derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol L |
| CN104447781A (en) * | 2014-12-18 | 2015-03-25 | 福州大学 | Citrinin compound penicitrinol N derived from penicillium citrinum as well as preparation method and application thereof |
| CN104478891A (en) * | 2014-12-18 | 2015-04-01 | 福州大学 | Penicillium citrinum sourced citrinin compound (penicitrinol O) as well as preparation method and application thereof |
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