CN107325087A - 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 PDF

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Publication number
CN107325087A
CN107325087A CN201610361240.3A CN201610361240A CN107325087A CN 107325087 A CN107325087 A CN 107325087A CN 201610361240 A CN201610361240 A CN 201610361240A CN 107325087 A CN107325087 A CN 107325087A
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compound
penicillium citrinum
extract
ibpt
malignant
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CN107325087B (en
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陈立
赵杨杨
王丙帅
周彤
张其清
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Fuzhou University
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Fuzhou University
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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
    • 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
    • C12P17/16—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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  • Organic Chemistry (AREA)
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  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

The invention discloses a kind of citrinin compounds dicitrinone D and its application in terms of malignant mela noma, 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 people's Human melanoma cell line A375, the research of malignant mela noma can be used for as malignant melanoma cell Proliferation Ability medicine or antineoplastic is prepared.

Description

Citrinin compounds dicitrinone D and its in terms of malignant mela noma Using
Technical field
The invention belongs to biomedicine field, and in particular to a kind of citrinin compounds dicitrinone D and its Application in terms of malignant mela noma.
Background technology
Citrinin be citrinin species in the organic compounds containing nitrogen that a class is produced by biological cometabolism, nature compared with It is many, 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 being protruded the most with antibacterial, antitumor activity.Natural knot Structure citrinin is the important sources of discovery lead compound in innovation drug research, has been applied to clinical citrinin medicine at present Through nearly hundred kinds.Research finds that it is mould that some marine fungis can produce the tangerine that structure is novel, activity is good during cometabolism Element, with good medicinal and industrialization prospect.
The present inventor studies and learnt, 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) crude extract of tunning has good cell inhibitory effect activity, and its active component is studied then. Research finds that shown citrinin compounds have anti-malignant mela noma activity, has not yet to see the compound pernicious to people black The report of pigment oncocyte A375 proliferation inhibition activity, therefore in the market is also there is not yet medicine related to this.
The content of the invention
It is an object of the invention to provide a kind of citrinin compounds dicitrinone D preparation sides for coming from Penicillium citrinum Application in terms of method and its suppression malignant melanoma cell propagation, the compound, which has, suppresses malignant melanoma cell propagation Effect, with anti-malignant mela noma 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, is comprised the following steps that：
（1）Fermenting and producing
Cultivate microorganism conventional method, take Penicillium citrinum (Penicillium citrinum) IBPT-5 is inoculated into PDA solids Cultivate 4 days, be then seeded into nutrient solution in 28 DEG C of incubators on slant medium, 28 DEG C of static gas wave refrigerators are obtained after 30 days Mycelium and zymotic fluid；The nutrient solution composition：Every liter of water containing the g of mannitol 20.0, the g of yeast extract 3.0, the g of maltose 20.0, The g of monosodium glutamate 10.0, glucose 10.0 g, KH2PO4 0.5 g、MgSO4The 0.3 g and g of NaCl 30.0；
（2）The acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth, zymotic fluid is extracted twice with the ethyl acetate of two volumes, extract subtracts Pressure distillation obtains the ethyl acetate extract of zymotic fluid to dry；
（3）The separation and purification of compound
The step（2）After obtained ethyl acetate extract is by 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol Silica gel chromatographic column gradient elution is depressurized for eluent, with dichloromethane：Methanol is eluent, further passes through pressurized silica gel post Chromatographic grade is eluted, and obtains dichloromethane：Methanol v/v=100:1 eluate, then pass through 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 85% acetonitrile contains 0.1% TFA, obtains shownization 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 is：CCTCC NO：M 2013713.
The present invention also protects described compound preparing the purposes in suppressing malignant melanoma cell hyperproliferation agent, And purposes of the compound in anti-malignant mela noma medicine is prepared, the melanoma cells behaviour malignant mela noma is thin Born of the same parents A375.
The beneficial effects of the present invention are：The citrinin compound shown in research is more rare, the citrinin class chemical combination Thing has significant suppression malignant melanoma cell proliferation activity, has not yet to see the compound thin to people's malignant mela noma The report of born of the same parents' A375 proliferation inhibition activities, therefore in the market is also there is not yet medicine related to this.
Brief description of the drawings
Fig. 1 is main COSY, HMBC and the NOE signals of Dicitrinone D.
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 constitution of signified compound in examples below：
。
The fermenting and producing and separation and purification of the compound of embodiment 1
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 Wuhan University, protects Hiding numbering is：CCTCC NO：M 2013713) in right amount, it is inoculated into PDA solid slope culture mediums and is cultivated in 28 DEG C of incubators 4 days.
Take the inclined-plane culture Penicillium citrinum of 4 days（Penicillium citrinum）Appropriate IBPT-5, is inoculated into equipped with 400mL [nutrient solution constitutes (g/l) to nutrient 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 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, and extract subtracts Pressure distillation obtains the g of ethyl acetate extract 32.0 of zymotic fluid to dry.
The separation and purification of 3 compounds
After the medicinal extract is by 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol is that eluent depressurizes silica gel color Post gradient elution is composed, 11 components are obtained.Component 3 (1.8 g) (eluate of dichloromethane) is with dichloromethane：Methanol is to wash De- liquid, further by pressurized silica gel column chromatography gradient elution, obtained subfraction 3-2 (210 mg) (dichloromethane：Methanol v/v=100:1 eluate), then pass through semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm)：Separate flow velocity For 5 mL/min, 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 exists m/z359.1457 place provide molecular ion peak [M+ H]+, (calcd. for C20H23O6, 359.1489), it is 358 to point out molecular weight, speculates that molecular formula is with reference to spectral information C20H22O6。1H and13C-NMR data are shown in Table 1, and main COSY, HMBC and NOE signal is 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 method
The preparation test sample of sample solution is the pure compounds of separation and purification in above-mentioned implementation 1.Precision is weighed in right amount Sample, the solution of concentration needed for being configured to 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 The culture mediums of RPMI 1640, 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 hole 100 μ L are inoculated in 96 porocyte culture plates, and 5% CO is passed through in 37 DEG C2Incubator in overnight incubation.Supernatant is sucked, addition contains The μ L of culture medium 100 of sample, continue to cultivate 48 h.10 μ L WST-1 liquid are added per hole, 4 h are cultivated.Utilize Bio-Rad companies 680 type ELIASAs are produced to determine per light absorption value (OD) value of hole at 450nm.Each concentration of sample is equal in the orifice plate of same 96 Five holes are set, five hole blank controls and acellular zeroing hole are separately set (if to have color to do relative medicine concentration acellular for medicine Zeroing).Each hole OD values first do corresponding acellular zeroing, then take 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 methods, according to the malignant melanoma cell proliferation inhibition rate of the compound of various concentrations, application SPSS16.0 softwares carry out data processing and calculation of half inhibitory concentration IC50Value.It the results are shown in Table 2.
The inhibitory activity that the compound of table 2 is bred to people's malignant melanoma cell
3. conclusion
The compound has preferable antitumor activity to people's Human melanoma cell line A375.Can be plain as maligna is prepared Tumor cell proliferation, which suppresses medicine or antineoplastic, is used for the research of malignant mela noma.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, should all belong to the covering scope of the present invention.

Claims (6)

1. compound。
2. a kind of preparation method of compound as claimed in claim 1, it is characterised in that：Fermented and cultured Penicillium citrinum IBPT-5, is obtained Fermentate is taken, the compound, the Penicillium citrinum IBPT-5, on December 25th, 2013 are then isolated and purified out from fermentate It is deposited in China typical culture collection center, address:Wuhan Wuhan University, deposit number is：CCTCC NO：M 2013713。
3. the preparation method of the compound according to right 2, it is characterised in that：Comprise the following steps that：
（1）Fermenting and producing
The conventional method of microorganism is cultivated, takes Penicillium citrinum IBPT-5 to be inoculated into PDA solid slope culture mediums in 28 DEG C of incubators Middle culture 4 days, is then seeded into nutrient solution, and 28 DEG C of static gas wave refrigerators obtain mycelium and zymotic fluid after 30 days；The nutrient solution Composition：Every liter of water containing the g of mannitol 20.0, the g of yeast extract 3.0, the g of maltose 20.0, the g of monosodium glutamate 10.0, the g of glucose 10.0, KH2PO4 0.5 g、MgSO4The 0.3 g and g of NaCl 30.0；
（2）The acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth, zymotic fluid is extracted twice with the ethyl acetate of two volumes, extract subtracts Pressure distillation obtains the ethyl acetate extract of zymotic fluid to dry；
（3）The separation and purification of compound
By step（2）After obtained ethyl acetate extract is by 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol Silica gel chromatographic column gradient elution is depressurized for eluent, with dichloromethane：Methanol is eluent, further passes through pressurized silica gel post Chromatographic grade is eluted, and obtains dichloromethane：Methanol v/v=100:1 eluate, then pass through the type of semi-preparative liquid chromatography 1010 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 Compound, tR 6.8 min。
4. the compound described in claim 1 is preparing the purposes in suppressing malignant melanoma cell hyperproliferation agent.
5. purposes of the compound in anti-malignant mela noma medicine is prepared described in claim 1.
6. purposes according to claim 4, it is characterised in that：Described melanoma cells are malignant melanoma cell A375。
CN201610361240.3A 2016-05-27 2016-05-27 Citrinin compounds dicitrinone D and its application in terms of malignant mela noma Expired - Fee Related CN107325087B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573568A (en) * 2020-12-02 2022-06-03 广州中医药大学(广州中医药研究院) 2-quinolinone-citrinin hybrid dimer compound and preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
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

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114573568A (en) * 2020-12-02 2022-06-03 广州中医药大学(广州中医药研究院) 2-quinolinone-citrinin hybrid dimer compound and preparation method and application thereof
CN114573568B (en) * 2020-12-02 2023-06-02 广州中医药大学(广州中医药研究院) 2-quinolinone-citrinin hybrid dimer compound and preparation method and application thereof

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