CN110407795A - Derived from penicillium oxalicum secalonic acid L and inhibiting the application in human cancer cell proliferation - Google Patents

Derived from penicillium oxalicum secalonic acid L and inhibiting the application in human cancer cell proliferation Download PDF

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CN110407795A
CN110407795A CN201910344822.4A CN201910344822A CN110407795A CN 110407795 A CN110407795 A CN 110407795A CN 201910344822 A CN201910344822 A CN 201910344822A CN 110407795 A CN110407795 A CN 110407795A
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penicillium oxalicum
cancer cell
human cancer
compound
cell proliferation
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CN110407795B (en
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刘沁颖
郑秋红
许扬梅
陈立
鲁志浩
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Fujian Cancer Hospital (fujian Cancer Institute Fujian Cancer Prevention And Treatment Center)
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Fujian Cancer Hospital (fujian Cancer Institute Fujian Cancer Prevention And Treatment Center)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
    • C07D311/86—Oxygen atoms, e.g. xanthones
    • 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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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

The present invention relates to a kind of derived from penicillium oxalicum secalonic acid L and in the application inhibited in human cancer cell proliferation.The compound, which has, inhibits human cancer cell proliferation function.Its structural formula are as follows:.By fermented and cultured penicillium oxalicum (Penicillium oxalicum) IBPT-6, 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 a variety of human cancer cells.Can be used for preparing cancer cell multiplication inhibits drug or anti-tumor drug for studying.

Description

Derived from penicillium oxalicum secalonic acid L and inhibiting the application in human cancer cell proliferation
Technical field
The present invention relates to a kind of derived from the secalonic acid class compound Secalonic acid L of penicillium oxalicum and its inhibition The application of human cancer cell proliferation aspect, belongs to biomedicine field.
Background technique
Secalonic acid class compound (Secalonic acids) belongs to the production of ergot pigment (Ergochrome) secondary metabolism Object is xanthone dimer.Since Stoll etc. was in the isolated secalonic acid A from fungi in 1952 After (Secalonic acid A), the compound secalonic acid (A-I) of the series is just constantly found and studies.Rye ketone Acid compounds have a variety of different physiological activity, with secalonic acid D(Secalonic acid D, SAD) for, 5 mg/ The SAD of ml is added in physiological saline, within the scope of 5-20 mg, it can treatment early stage bladder cancer, 50-100 mg range It is interior, and be free from side effects generation effective in cure to more serious bladder cancer.It has been investigated that some marine fungis can be in secondary The good secalonic acid class compound of structure novel, activity is generated in metabolic process, and there is good medicinal and industrialization prospect.
The present inventor studies and learns, penicillium oxalicum (Penicillium oxalicum) IBPT-6 has been (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 2013714) crude extract of tunning has good cell inhibitory effect activity, studies then its active constituent.It grinds Studying carefully secalonic acid class compound shown in discovery has anti-human cancer activity, has not yet to see the compound and presses down to the proliferation of human cancer cell Active report is made, 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 secalonic acid class compound Secalonic acid derived from penicillium oxalicum L and its application for inhibiting human cancer cell proliferation aspect.The compound, which has, inhibits cancer cell multiplication effect, has anticancer activity. Its structural formula are as follows:
。
The preparation method of the compound, be by fermented and cultured penicillium oxalicum (Penicillium oxalicum) IBPT-6 obtains fermentation material, the compound is then isolated and purified out from fermentation material.Specific step is as follows:
1 fermenting and producing
Cultivate microorganism conventional method, take penicillium oxalicum (Penicillium oxalicum) to be inoculated into PDA solid by IBPT-6 It cultivates 2 to 3 days, is then seeded into culture solution in 28 DEG C of incubators on body slant 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、MgSO4·7H2O 0.3 g、NaCl 15.0 g。
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.Mycelium with acetone soln (contain 20% ~ 30% water) continuous ultrasound broken wall 3 times, Filtering removal residue, obtains the mycelium crude extract containing acetone and water.Removal acetone is concentrated under reduced pressure, obtains the water-soluble of crude extract Liquid, then ethyl acetate is added with volume ratio 1:2 and is extracted 3 times, ethyl acetate crude extract is obtained, is concentrated under reduced pressure into close dry that mycelium soaks 36.5 g of cream.
The separation and purification of 3 compounds
Mycelium medicinal extract is by 100-200 mesh silica gel mixed sample, and using petroleum ether: methylene chloride: methanol is subtracted as gradient eluent Press silica gel chromatographic column chromatography.By simple thin-layer chromatographic analysis, merges, be separated into component A-E.Component D (5.9 g) (two Chloromethanes: the eluate of methanol v/v=100:1) using methylene chloride: methanol carries out pressured column silica gel as gradient elution agent Analysis merges after thin-layer chromatographic analysis and obtains five subfraction D1-D5.Component D3(1.1 g) with methanol: sour water (contains 0.1% Trifluoroacetic acid) it is that gradient elution agent carries out reversed chromatographed on silica gel, isolated four subfraction D3-1 ~ D3-4.The Asia of D3 Component D3-2(168 mg) it is separated under the conditions of mobile phase is 50% acetonitrile (containing 0.1% trifluoroacetic acid) by semi-preparative liquid chromatography Obtain the compound (2.1 mg).
The penicillium oxalicum (Penicillium oxalicum) IBPT-6, it has been deposited on December 25th, 2013 State's Type Tissue Collection, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013714.
The present invention also protects the compound to inhibit application and the chemical combination in human cancer cell hyperproliferation agent in preparation Object is preparing the application in anti-human cancer drug.
Remarkable advantage of the invention: the secalonic acid compound shown in studying has not been reported and has significant inhibition cancer thin Born of the same parents' proliferation activity has not yet to see the compound to the report of cancer cell multiplication inhibitory activity, thus in the market also there is not yet Drug related to this.
Detailed description of the invention
Fig. 1 Secalonic acid main COSY of L, HMBC and NOE signal.
Specific embodiment
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 oxalicum (Penicillium oxalicum) IBPT-6 (be deposited in China typical culture collection center on December 25th, 2013, address: protect by Wuhan Wuhan University Hiding number is: CCTCC NO:M 2013714) in right amount, being inoculated into PDA solid slope culture medium and cultivates in 28 DEG C of incubators 2 to 3 days.
Take inclined-plane culture 2 to 3 days penicillium oxalicum (Penicillium oxalicum) appropriate IBPT-6, it is inoculated into dress By 400mL culture solution, [culture solution is formed (grams per liter): mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, Portugal Grape sugar 10.0, KH2PO40.5, MgSO40.3, NaCl 15.0, constant volume] 1000mL conical flask in, 28 DEG C of static gas wave refrigerators After 30 days, mycelium and fermentation liquid are obtained.
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.Mycelium with acetone soln (contain 20% ~ 30% water) continuous ultrasound broken wall 3 times, Filtering removal residue, obtains the mycelium crude extract containing acetone and water.Removal acetone is concentrated under reduced pressure, obtains the water-soluble of crude extract Liquid, then ethyl acetate is added with volume ratio 1:2 and is extracted 3 times, ethyl acetate crude extract is obtained, is concentrated under reduced pressure into close dry that mycelium soaks 36.5 g of cream.
The separation and purification of 3 compounds
Mycelium medicinal extract is by 100-200 mesh silica gel mixed sample, and using petroleum ether: methylene chloride: methanol is subtracted as gradient eluent Press silica gel chromatographic column chromatography.By simple thin-layer chromatographic analysis, merges, be separated into component A-E.Component D (5.9 g) (two Chloromethanes: the eluate of methanol v/v=100:1) using methylene chloride: methanol carries out pressured column silica gel as gradient elution agent Analysis merges after thin-layer chromatographic analysis and obtains five subfraction D1-D5.Component D3(1.1 g) with methanol: sour water (contains 0.1% Trifluoroacetic acid) it is that gradient elution agent carries out reversed chromatographed on silica gel, isolated four subfraction D3-1 ~ D3-4.The Asia of D3 Component D3-2(168 mg) it is separated under the conditions of mobile phase is 50% acetonitrile (containing 0.1% trifluoroacetic acid) by semi-preparative liquid chromatography Obtain the compound (2.1mg).
Compound: being yellow oil under room temperature, and high-resolution electrospray ionization mass spectrum HRESI-MS gives at m/z:661.1537 Molecular ion peak [M+Na]+(calcd for C out32H30NaO14, 661.1533), molecular ion peak [M-is provided at 637.1568 H]-(calcd for C32H29O14, 637.1557), infer that its molecular formula is C32H30O14。1H and13C-NMR data are shown in Table 1, master COSY, HMBC and NOE signal wanted are shown in Fig. 1.
1 compound of table1H and13C-NMR data (500MHz1H and 125 MHz 13C, in DMSO d6)
ab Signal can be interchanged.
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-described embodiment 1.Precision weighs suitable Sample is measured, the solution of required concentration is configured to methanol, for surveying activity.
The squamous subculture of cell line and cell uses tumor cell line, and tumour cell uses the DMEM containing 10% FBS to cultivate Base, at 37 DEG C in being passed through 5% CO2Incubator in squamous subculture.
Cell inhibitory effect activity test method
Tetrazolium (MTT) method: cell density is adjusted to every milliliter 2 × 10 by the tumour cell of logarithmic growth phase5A cell, It is inoculated in 96 porocyte culture plates for 200 microlitres by every hole, is passed through 5% CO in 37 DEG C2Incubator in cultivate 4 hours.Every hole 2 microlitres of sample liquid or blank solution is added, after culture 24 hours, MTT liquid (every milliliter of 5 milligrams of physiology of MTT are added in every hole Saline solution) 10 microlitres, continue culture 4 hours, 37 DEG C, 2000 revs/min are centrifuged 8 minutes, draw supernatant.DMSO is added in every hole It each 100 microlitres, is vibrated 15 minutes on micro oscillator, until utilizing the production of MD company after crystallization is completely dissolved SPECTRAMAX Plus type microplate reader measures extinction (OD) value of every hole at 570 nm.The sample in 96 orifice plate of same Each concentration is respectively provided with three holes, and the another blank control that three holes are arranged and cell-free withered hole are (if drug has color to do accordingly It is withered that drug concentration is cell-free).Each hole OD value first do it is corresponding cell-free withered, then take three hole mean OD values by IR (%)= (ODBlank control-ODSample)/ODBlank control× 100% calculates the proliferation inhibition rate (IR%) of cell under each concentration.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, according to the Cytostatic to tumor cell rate of the compound of various concentration, using SPSS16.0 software Carry out data processing and calculation of half inhibitory concentration IC50Value.It the results are shown in Table 2.
Inhibitory activity (IC of 2 compound of table to cancer cell multiplication50, μM)
3. conclusion
The compound has preferable anti-tumor activity to human cancer cell.It can be used as and prepare cancer cell multiplication inhibition drug or anti-swollen Tumor medicine is used for the research of 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 (3)

1. compound。
2. compound described in claim 1 inhibits the application in human cancer cell hyperproliferation agent in preparation.
3. compound described in claim 1 is preparing the application in anti-human cancer drug.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4424373A (en) * 1979-01-30 1984-01-03 Asahi Kasei Kogyo Kabushiki Kaisha Secalonic acids
CN109106702A (en) * 2017-12-19 2019-01-01 福州大学 Derived from application of 4-4 ' the isomerization secalonic acid D in terms of colon cancer of penicillium oxalicum

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4424373A (en) * 1979-01-30 1984-01-03 Asahi Kasei Kogyo Kabushiki Kaisha Secalonic acids
CN109106702A (en) * 2017-12-19 2019-01-01 福州大学 Derived from application of 4-4 ' the isomerization secalonic acid D in terms of colon cancer of penicillium oxalicum

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ANTONIUS R. B. OLA ET AL.: ""Absolute configuration and antibiotic activity of neosartorin from the endophytic fungus Aspergillus fumigatiaffinis"", 《TETRAHEDRON LETTERS》 *
LI CHEN ET AL.: ""Secalonic acids J-M, four new secondary metabolites from the marine-derived fungus Penicillium oxalicum"", 《HETEROCYCLES》 *
WEI FANG ET AL.: ""Cytotoxic and Antibacterial Eremophilane Sesquiterpenes from the Marine-Derived Fungus Cochliobolus lunatus SCSIO41401"", 《JOURNAL OF NATURAL PRODUCTS》 *
WEN ZHANG ET AL.: ""New Mono- and Dimeric Members of the Secalonic Acid Family: Blennolides A–G Isolated from the Fungus Blennoria sp."", 《CHEM. EUR. J.》 *
YUDAI MATSUDA ET AL.: ""Genetic Characterization of Neosartorin Biosynthesis Provides Insight into Heterodimeric Natural Product Generation"", 《ORGANIC LETTERS》 *
李庆 等: ""真菌代谢产物Secalonic acid D及其药理活性研究"", 《中药材》 *

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