CN105001228A - Application of penicillium citrinum-derived penicitrinine A in preparing drugs for treating human oral epidermoid tumor - Google Patents
Application of penicillium citrinum-derived penicitrinine A in preparing drugs for treating human oral epidermoid tumor Download PDFInfo
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- CN105001228A CN105001228A CN201510423834.8A CN201510423834A CN105001228A CN 105001228 A CN105001228 A CN 105001228A CN 201510423834 A CN201510423834 A CN 201510423834A CN 105001228 A CN105001228 A CN 105001228A
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- penicitrinine
- oral epidermoid
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- C—CHEMISTRY; METALLURGY
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- 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
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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
- C12P17/18—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
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Abstract
The invention discloses an alkaloidal compound penicitrinine A derived from penicillium citrinum, and the application of the penicillium citrinum-derived penicitrinine A in preparing drugs for inhibiting the cell proliferation of human oral epidermoid tumor cells or antitumor drugs. The structural formula of the penicitrinine A is shown in img file='dest_path_image002.TIF' wi='251' he='135'. After the fermental cultivation of penicillium citrinum ((i) Penicillium (/i)(i) citrinum (/i)) IBPT-5, fermentation products are obtained, separated and purified to obtain the above compound. The results of experiments show that, the compound is better in antitumor activity for human oral epidermoid tumor cells KB. Therefore, the compound can be used for preparing drugs for inhibiting the cell proliferation of human oral epidermoid tumor cells or antitumor drugs.
Description
Technical field
The invention belongs to antitumor drug preparation field, be specifically related to a kind of come from Penicillium citrinum alkaloid compound penicitrinine A and application in preparation human oral epidermoid cytostatic thing or antitumor drug.
Background technology
Alkaloid is the organic compounds containing nitrogen that a class is produced by biological secondary metabolism, and the alkaloid kind of occurring in nature is more, mostly derives from plant, therefore has again the title of vegeto-alkali.Alkaloid has important physiological action to humans and animals, comprises antiasthmatic effect, hypoglycemic, reducing blood-fat, antibacterial, antitumor, analgesia etc., wherein with antibacterial, anti-tumor activity is the most outstanding.Natural structure alkaloid is the important sources finding lead compound in innovation drug research, is applied to clinical alkaloidal drug nearly hundred kinds at present.Research finds, some thalassiomycetess can produce novel structure, active good alkaloid in secondary metabolism process, have well medicinal and industrialization prospect.
The present inventor's research is learnt, Penicillium citrinum (
penicillium citrinum) IBPT-5, (be deposited in China typical culture collection center on December 25th, 2013, address: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2013713) the crude extract of tunning have good cell inhibitory effect active, then its activeconstituents is studied.It is active that alkaloid compound shown in research finds has anti-human oral epidermoid carcinoma, has not yet to see the report of this compound to the proliferation inhibition activity of human oral epidermoid cell KB, therefore market also there is not yet medicine related to this.
Summary of the invention
The object of the present invention is to provide a kind of come from Penicillium citrinum alkaloid compound penicitrinine A and application in preparation human oral epidermoid cytostatic thing or antitumor drug.This compound has suppression human oral epidermoid cel l proliferation, has anti-human oral epidermoid carcinoma active.
The structural formula of this compound is:
。
The preparation method of this compound be by fermentation culture Penicillium citrinum (
penicillium citrinum) IBPT-5, obtain fermented product, then from fermented product, separation and purification goes out this compound.Concrete steps are as follows:
1, fermentative production
The ordinary method of culturing micro-organisms, get Penicillium citrinum (
penicillium citrinum) IBPT-5 to be inoculated on PDA solid slant culture base and to cultivate 4 days in 28 DEG C of incubators, be then inoculated in nutrient solution, 28 DEG C of static gas wave refrigerator, after 30 days, obtain mycelium and fermented liquid; Described nutrient solution composition: every premium on currency is containing N.F,USP MANNITOL 20.0 g, yeast extract paste 3.0 g, maltose 20.0 g, monosodium glutamate 10.0 g, glucose 10.0 g, KH
2pO
40.5 g, MgSO
40.3 g, NaCl 30.0 g;
2, the acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth.By fermented liquid ethyl acetate 1:2 (v/v) extracting twice, extraction liquid underpressure distillation, to dry, obtains ethyl acetate extract 32.0 g of fermented liquid.
3, the separation and purification of compound
After this medicinal extract passes through 100-200 order silica gel mixed sample, with sherwood oil: methylene dichloride: methyl alcohol is elutriant decompression silica gel chromatographic column gradient elution, obtains 11 components.Component 7 (4.2 g) (methylene dichloride: the eluate of methyl alcohol v/v=100:1) with methylene dichloride: methyl alcohol is elutriant, further by pressurized silica gel column chromatography gradient elution, the subfraction 7-6 (methylene dichloride: the eluate of methyl alcohol v/v=50:1) obtained is by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm): being separated flow velocity is 5 mL/min, moving phase is that 75% acetonitrile is containing 0.1% TFA, obtain shown compound (97.9 mg, t
r19.3 min).
Described Penicillium citrinum (Penicillium citrinum) IBPT-5, be deposited in China typical culture collection center, address on December 25th, 2013: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2013713.
This compound suppresses the purposes in human oral epidermoid cell proliferation in preparation, and the purposes of this compound in the anti-human oral epidermoid carcinoma medicine of preparation.Described tumour cell behaviour oral epidermoid carcinoma cell KB.
Remarkable advantage of the present invention is: shown in research, this alkaloid compound is comparatively rare, described alkaloid compound has significant suppression human oral epidermoid cell-proliferation activity, have not yet to see the report of this compound to human oral epidermoid cell KB proliferation inhibition activity, therefore market also there is not yet medicine related to this.
Accompanying drawing explanation
Fig. 1 is the main COSY of Penicitrinine A, HMBC and NOESY signal.
Embodiment
The chemical structure of the compound of indication in following embodiment:
。
The fermentative production of this compound of embodiment 1 and separation and purification
1, fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, get Penicillium citrinum (
penicillium citrinum) IBPT-5 (is deposited in China typical culture collection center on December 25th, 2013, address: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2013713) appropriate, be inoculated on PDA solid slant culture base and cultivate 4 days in 28 DEG C of incubators.
Get the slant culture Penicillium citrinum of 4 days (
penicillium citrinum) IBPT-5 is appropriate, be inoculated into and 400mL nutrient solution [nutrient solution composition (grams per liter): N.F,USP MANNITOL 20.0, yeast extract paste 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0, KH is housed
2pO
40.5, MgSO
40.3, NaCl 30.0 constant volume] 1000mL Erlenmeyer flask in, 28 DEG C of static gas wave refrigerator are after 30 days, obtain mycelium and fermented liquid.
2, the acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth.By fermented liquid ethyl acetate 1:2 (v/v) extracting twice, extraction liquid underpressure distillation, to dry, obtains ethyl acetate extract 32.0 g of fermented liquid.
3, the separation and purification of compound
After this medicinal extract passes through 100-200 order silica gel mixed sample, with sherwood oil: methylene dichloride: methyl alcohol is elutriant decompression silica gel chromatographic column gradient elution, obtains 11 components.Component 7 (4.2 g) (methylene dichloride: the eluate of methyl alcohol v/v=100:1) with methylene dichloride: methyl alcohol is elutriant, further by pressurized silica gel column chromatography gradient elution, the subfraction 7-6 (methylene dichloride: the eluate of methyl alcohol v/v=50:1) obtained is by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm): being separated flow velocity is 5 mL/min, moving phase is that 75% acetonitrile is containing 0.1% TFA, obtain shown compound (97.9 mg, t
r19.3 min).
Compound is yellow oily, and high resolution mass spectrum HRESI-MS exists
m/z484.2711 places provide molecular ion peak [M – H]
–, (calcd. for C
28h
38nO
6, 484.2705), prompting molecular weight is 485, infers that molecular formula is C in conjunction with spectral information
28h
39nO
6.
1h and
13c-NMR data are in table 1, and main COSY, HMBC and NOESY signal is shown in Fig. 1.
Table 1 NMR compound
1h and
13c-NMR data (500MHz
1h and 125MHz
13c, in CDCl
3)
The test of embodiment 2 anti tumor activity in vitro
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 takes appropriate amount of sample, is mixed with the solution of desired concn with DMSO, active for survey.
The succeeding transfer culture of clone and cell adopts tumor cell line, and RPMI 1640 substratum of tumour cell containing 10% FBS, at 37 DEG C in passing into 5% CO
2incubator in succeeding transfer culture.
Cell inhibitory effect activity test method
The tumour cell that WST-1 method is taken the logarithm vegetative period, is adjusted to every milliliter 5 × 10 by cell density
4individual cell, is inoculated in 96 porocyte culture plates by every hole 100 μ L, passes into 5% CO in 37 DEG C
2incubator in overnight incubation.Suck supernatant liquor, add the substratum 100 μ L containing sample, continue cultivation 48 h.Every hole adds 10 μ L WST-1 liquid, cultivates 4 h.Utilize Bio-Rad company to produce 680 type microplate reader and measure light absorption value (OD) value of every hole at 450nm place.In same 96 orifice plate, each concentration of sample all arranges five holes, separately establishes five hole blanks and acellular zeroing hole (if medicine has color will do the acellular zeroing of relative medicine concentration).Each hole OD value first does corresponding acellular zeroing, then gets five hole mean OD value by IR (%)=(OD
blank-OD
sample)/OD
blank× 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 Cytostatic to tumor cell rate of this compound of different concns, application SPSS16.0 software carries out data processing and calculation of half inhibitory concentration IC
50value.The results are shown in Table 2.
Table 2 compound is to the inhibit activities of human oral epidermoid cell proliferation
3. conclusion
This compound has good anti-tumor activity to human oral epidermoid cell KB.Can be used as preparation human oral epidermoid cytostatic thing or antitumor drug for the research of human oral epidermoid.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. come from the alkaloid compound penicitrinine A of Penicillium citrinum, it is characterized in that: structural formula is as follows:
.
2. the application of compound as claimed in claim 1 in preparation suppression human oral epidermoid cell proliferation.
3. the application of compound as claimed in claim 1 in the anti-human oral epidermoid carcinoma medicine of preparation.
4. application according to claim 2, is characterized in that: tumour cell behaviour oral epidermoid carcinoma cell KB.
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| CN105001228B CN105001228B (en) | 2017-03-22 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004000333A1 (en) * | 2002-06-20 | 2003-12-31 | Astion Dermatology A/S | Novel complexes of fatty acid esters of polyhydroxyalkanes and pyridine carboxy derivatives |
| WO2006075395A1 (en) * | 2005-01-11 | 2006-07-20 | The Kitasato Institute | β-LACTAM ANTIBIOTIC ACTIVITY ENHANCER AND PROCESS FOR PRODUCING THE SAME |
| 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 |
| CN104592082A (en) * | 2014-12-18 | 2015-05-06 | 福州大学 | Method for preparing penicillium enol D2 derived from Penicillium citrinum and application of penicillium enol D2 |
-
2015
- 2015-07-17 CN CN201510423834.8A patent/CN105001228B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004000333A1 (en) * | 2002-06-20 | 2003-12-31 | Astion Dermatology A/S | Novel complexes of fatty acid esters of polyhydroxyalkanes and pyridine carboxy derivatives |
| WO2006075395A1 (en) * | 2005-01-11 | 2006-07-20 | The Kitasato Institute | β-LACTAM ANTIBIOTIC ACTIVITY ENHANCER AND PROCESS FOR PRODUCING THE SAME |
| 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 |
| CN104592082A (en) * | 2014-12-18 | 2015-05-06 | 福州大学 | Method for preparing penicillium enol D2 derived from Penicillium citrinum and application of penicillium enol D2 |
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Granted publication date: 20170322 Termination date: 20180717 |