CN105153175A - Penicitrinine A derived from penicillium citrinum and application of penicitrinine A in preparing anti-human esophagus cancer drug - Google Patents

Penicitrinine A derived from penicillium citrinum and application of penicitrinine A in preparing anti-human esophagus cancer drug Download PDF

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CN105153175A
CN105153175A CN201510422123.9A CN201510422123A CN105153175A CN 105153175 A CN105153175 A CN 105153175A CN 201510422123 A CN201510422123 A CN 201510422123A CN 105153175 A CN105153175 A CN 105153175A
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compound
esophagus cancer
human esophagus
penicitrinine
penicillium citrinum
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CN105153175B (en
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郑秋红
刘沁颖
陈立
应敏刚
周彤
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FUJIAN CANCER HOSPITAL
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FUJIAN CANCER HOSPITAL
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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
    • 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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  • Engineering & Computer Science (AREA)
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  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
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  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

The invention relates to penicitrinine A derived from penicillium citrinum and application of penicitrinine A in preparing an anti-human esophagus cancer drug. The compound plays a role of inhibiting cell proliferation of human esophagus cancer. The structural formula of the compound is shown in the description. The compound is prepared by the steps: carrying out fermental cultivation of Penicillium citrinum IBPT-5 to obtain a fermented substance; and then separating purifying the compound from the fermented substance. Experiments show that the compound has relatively good anti-tumor activity on human esophagus cancer cells EC-9706 and KYSE450 and can be used for preparing anti-human esophagus cancer proliferation inhibiting drugs or anti-tumor drugs for research of human esophagus cancer.

Description

Come from the penicitrinine A of Penicillium citrinum and prepare the application of anti-human esophageal carcinoma medicine
Technical field
The present invention relates to and a kind ofly come from the penicitrinineA of Penicillium citrinum and prepare the application of anti-human esophageal carcinoma medicine.
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 ( penicilliumcitrinum) IBPT-5, (be deposited in China typical culture collection center on December 25th, 2013, address: Wuhan Wuhan University, deposit number is: CCTCCNO:M2013713) 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 the anti-human esophageal carcinoma, has not yet to see the report of this compound to the proliferation inhibition activity of human esophagus cancer cell EC-9706, KYSE450, therefore market also there is not yet medicine related to this.
Summary of the invention
The object of the present invention is to provide and a kind ofly come from the penicitrinineA of Penicillium citrinum and prepare the application of anti-human esophageal carcinoma medicine.This compound has suppression human esophagus cancer cell proliferation function, has the anti-human esophageal carcinoma active.Its structural formula is:
。
The preparation method of this compound, be by fermentation culture Penicillium citrinum ( penicilliumcitrinum) 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 ( penicilliumcitrinum) 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 20.0g, yeast extract paste 3.0g, maltose 20.0g, monosodium glutamate 10.0g, glucose 10.0g, KH 2pO 40.5g, MgSO 40.3g, NaCl30.0g;
The acquisition of 2 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 the ethyl acetate extract 32.0g of fermented liquid.
The separation and purification of 3 compounds
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 (4.2g) (methylene dichloride: the eluate of methyl alcohol v/v=100:1) is 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 × 250mm, 5 μm): separation flow velocity is 5mL/min, moving phase is that 75% acetonitrile is containing 0.1%TFA, obtain shown compound (97.9mg, t r19.3min).
Described Penicillium citrinum (Penicilliumcitrinum) IBPT-5, is deposited in China typical culture collection center, address on December 25th, 2013: Wuhan Wuhan University, deposit number is: CCTCCNO:M2013713.
Described compound suppresses the purposes in human esophagus cancer cell hyperproliferation agent in preparation, and the purposes of this compound in the anti-human esophageal carcinoma medicine of preparation.Described tumour cell is human esophagus cancer cell EC-9706, KYSE450.
Remarkable advantage of the present invention: shown in research, this alkaloid compound is comparatively rare, described alkaloid compound has significant suppression human esophagus cancer cell proliferation activity, have not yet to see the report of this compound to human esophagus cancer cell EC-9706, KYSE450 proliferation inhibition activity, therefore market also there is not yet medicine related to this.
Accompanying drawing explanation
The COSY that Fig. 1 PenicitrinineA is main, 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 ( penicilliumcitrinum) IBPT-5 (is deposited in China typical culture collection center on December 25th, 2013, address: Wuhan Wuhan University, deposit number is: CCTCCNO:M2013713) 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 ( penicilliumcitrinum) IBPT-5 be inoculated into be equipped with 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 2pO 40.5, MgSO 40.3, NaCl30.0 constant volume] 1000mL Erlenmeyer flask in, 28 DEG C of static gas wave refrigerator are after 30 days, obtain mycelium and fermented liquid.
The acquisition of 2 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 the ethyl acetate extract 32.0g of fermented liquid.
The separation and purification of 3 compounds
After this medicinal extract passes through 200 order silica gel mixed samples, with sherwood oil: methylene dichloride: methyl alcohol is elutriant decompression silica gel chromatographic column gradient elution, obtains 11 components.Component 7 (4.2g) (methylene dichloride: the eluate of methyl alcohol v/v=100:1) is 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 × 250mm, 5 μm): separation flow velocity is 5mL/min, moving phase is that 75% acetonitrile is containing 0.1%TFA, obtain shown compound (97.9mg, t r19.3min).
Compound as yellow oily, high resolution mass spectrum HRESI-MS exists m/z484.2711 places provide molecular ion peak [M – H] –, (calcd.forC 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 1NMR compound 1h and 13c-NMR data (500MHz 1hand125MHz 13c, inCDCl 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 the RPMI1640 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 to cultivate 48h.Every hole adds 10 μ LWST-1 liquid, cultivates 4h.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.
The inhibit activities that table 2 compound is bred human esophagus cancer cell
3. conclusion
This compound has good anti-tumor activity to human esophagus cancer cell EC-9706, KYSE450.Can be used as and prepare the research for human esophagus cancer of human esophagus cancer cell Proliferation Ability medicine or antitumor drug.
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 (5)

1. come from the penicitrinineA of Penicillium citrinum, it is characterized in that: described structural formula of compound is .
2. the preparation method of compound as claimed in claim 1, is characterized in that: fermentation culture Penicillium citrinum ( penicilliumcitrinum) IBPT-5, obtain fermented product, then from fermented product, separation and purification goes out this compound, described Penicillium citrinum ( penicilliumcitrinum) IBPT-5, be deposited in China typical culture collection center, address on December 25th, 2013: Wuhan Wuhan University, deposit number is: CCTCCNO:M2013713.
3. compound as claimed in claim 1 suppresses the application in human esophagus cancer cell hyperproliferation agent in preparation.
4. application according to claim 3, is characterized in that: described tumour cell is human esophagus cancer cell EC-9706, KYSE450.
5. the application of compound as claimed in claim 1 in the anti-human esophageal carcinoma medicine of preparation.
CN201510422123.9A 2015-07-17 2015-07-17 The penicitrinine A for coming from Penicillium citrinum and its application for preparing anti-human oesophagus cancer drug Expired - Fee Related CN105153175B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
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
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
CN105061446A (en) * 2015-07-17 2015-11-18 福建省肿瘤医院 Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting nasopharyngeal carcinoma

Patent Citations (3)

* Cited by examiner, † Cited by third party
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
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
CN105061446A (en) * 2015-07-17 2015-11-18 福建省肿瘤医院 Penicillium citrinum-derived penicitrinine A as well as application thereof to preparation of drugs for resisting nasopharyngeal carcinoma

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