CN107325081B - Citrinin compounds dicitrinone D preparation method and its application in terms of lung cancer - Google Patents
Citrinin compounds dicitrinone D preparation method and its application in terms of lung cancer Download PDFInfo
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- CN107325081B CN107325081B CN201610361188.1A CN201610361188A CN107325081B CN 107325081 B CN107325081 B CN 107325081B CN 201610361188 A CN201610361188 A CN 201610361188A CN 107325081 B CN107325081 B CN 107325081B
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom 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 preparation method and its application in terms of lung cancer, 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 good anti-tumor activity to human lung carcinoma cell SPC-A1, can be used as preparation proliferation of lung cancer cells and drug or anti-tumor drug is inhibited to be used for the research of lung cancer.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of preparation side citrinin compounds dicitrinone D
Method and its application in terms of lung carcinoma cell.
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-lung cancer activity, has not yet to see the compound to human lung carcinoma cell SPC-A1
Proliferation inhibition activity report, 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
Application in terms of method and its inhibition proliferation of lung cancer cells, which, which has, inhibits proliferation of lung cancer cells effect, has anti-lung cancer
Activity, structural formula are as follows:
。
The preparation method of the compound is 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 purposes and the chemical combination in proliferation of lung cancer cells drug in preparation
Object is preparing the purposes in anti-lung-cancer medicament；The lung carcinoma cell is SPC-A1 cell.
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 proliferation of lung cancer cells activity, and it is living to SPC-A1 cell Proliferation Ability to have not yet to see the compound
The report of property, 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 lung carcinoma cell uses the RPMI 1640 containing 10% FBS to train
Base is supported, 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 proliferation of lung cancer cells inhibiting rate of the compound of various concentration, application
SPSS16.0 software carries out data processing and calculation of half inhibitory concentration IC50Value, the results are shown in Table 2.
The inhibitory activity that 2 compound of table is proliferated human lung carcinoma cell
3. conclusion
The compound has preferable anti-tumor activity to human lung carcinoma cell SPC-A1, can be used as preparation proliferation of lung cancer cells
Drug or anti-tumor drug is inhibited to be used for the research of lung cancer.
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 (5)
1. compound, it is characterised in that: structural formula is。
2. a kind of preparation method of compound as described in claim 1, it is characterised in that: fermented and cultured Penicillium citrinum IBPT-5 is obtained
Fermentation material is taken, the compound, the Penicillium citrinum IBPT-5, on December 25th, 2013 are then isolated and purified out from fermentation material
Be deposited in China typical culture collection center, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M
2013713；Specific step is as follows:
(1) fermenting and producing
The conventional method for cultivating microorganism, takes Penicillium citrinum IBPT-5 to be inoculated into PDA solid slope culture medium in 28 DEG C of incubators
Middle culture 4 days, is then seeded into culture solution, and 28 DEG C after static gas wave refrigerator 30 days, obtain mycelium and fermentation liquid；The culture solution
Composition: every liter of water containing 20.0 g of mannitol, 3.0 g of yeast extract, 20.0 g of maltose, 10.0 g of monosodium glutamate, 10.0 g of glucose,
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, extract liquor subtracts
Pressure distillation obtains the ethyl acetate extract of fermentation liquid to doing；
(3) separation and purification of compound
After the ethyl acetate extract that step (2) is obtained passes through 100-200 mesh silica gel mixed sample, with petroleum ether: methylene chloride: methanol
Silica gel chromatographic column gradient elution is depressurized for eluent, using methylene chloride: methanol further passes through pressurized silica gel column as eluent
Chromatographic grade elution, obtains methylene chloride: the eluate of methanol v/v=100:1, then passes through 1010 type of semi-preparative liquid chromatography
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 shownization
Close object, tR 6.8 min。
3. compound described in claim 1 inhibits the purposes in proliferation of lung cancer cells drug in preparation.
4. compound described in claim 1 is preparing the purposes in anti-lung-cancer medicament.
5. purposes according to claim 3, it is characterised in that: the lung carcinoma cell is people's SPC-A1 cell.
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| CN201610361188.1A CN107325081B (en) | 2016-05-27 | 2016-05-27 | Citrinin compounds dicitrinone D preparation method and its application in terms of lung cancer |
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| CN201610361188.1A CN107325081B (en) | 2016-05-27 | 2016-05-27 | Citrinin compounds dicitrinone D preparation method and its application in terms of lung cancer |
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| CN107325081A CN107325081A (en) | 2017-11-07 |
| CN107325081B true CN107325081B (en) | 2019-07-09 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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
- 2016-05-27 CN CN201610361188.1A patent/CN107325081B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| 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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