CN103058971A - Benzoquinone spiro compound derived from aspergillus aculeatus and application of benzoquinone spiro compound - Google Patents
Benzoquinone spiro compound derived from aspergillus aculeatus and application of benzoquinone spiro compound Download PDFInfo
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- CN103058971A CN103058971A CN2013100164721A CN201310016472A CN103058971A CN 103058971 A CN103058971 A CN 103058971A CN 2013100164721 A CN2013100164721 A CN 2013100164721A CN 201310016472 A CN201310016472 A CN 201310016472A CN 103058971 A CN103058971 A CN 103058971A
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- benzoquinone
- spiro compound
- compound
- aspergillus aculeatus
- cell
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Abstract
The invention relates to a benzoquinone spiro compound derived from aspergillus aculeatus and application of the benzoquinone spiro compound. The structural formula of the compound is shown in the specification. Experiments prove that the benzoquinone spiro compound has good antitumor activity. The benzoquinone spiro compound can be used for preparing cell proliferation inhibition drugs or antitumor drugs for antitumor study.
Description
Technical field
The present invention relates to a kind of quinone derivatives and application thereof that comes from microorganism Aspergillus aculeatus.
Background technology
Microorganism Aspergillus aculeatus (
Aspergillus aculeatus) be the source of many bioactive compoundss, the chemical research of microorganism Aspergillus aculeatus has been disclosed its secondary metabolite before preferably antibiotic, antimycotic, antiviral, desinsection, antitumor isoreactivity have been arranged, these active compounds can be divided into the peptide class by its type of architecture, alkaloids, sesquiterpenoids, fatty acid, xanthone, however benzoquinones rarely has report with phenyl ring in the literature by the structure that oxo bridge links to each other.The inventor studies and learns, microorganism Aspergillus aculeatus (
Aspergillus aculeatus) IBPT-3 (was deposited in Chinese Typical Representative culture collection center on December 24th, 2012, deposit number is: the crude extract of tunning CCTCC NO:M 2012544) has good cell inhibitory effect active, then its activeconstituents is studied.Benzoquinone compound had anti-tumor activity shown in research was found, had not yet to see the report of chemical structure and the cell inhibitory effect activity of this compound, so also there is not yet relevant therewith medicine on the market.
Summary of the invention
The object of the present invention is to provide a kind of quinone derivatives and application thereof that comes from microorganism Aspergillus aculeatus.This compound has the inhibition tumor cell proliferation function, has anti-tumor activity.Its structural formula is:
。
Its constitutional features is: contain that four methyl all are in contraposition, the unparalleled key of a side in the benzoquinones in the molecular skeleton, molecule of the spirane structure that a benzoquinones and phenyl ring link to each other with oxo bridge by carbonyl.
The present invention has also protected the purposes of described compound in preparation cytostatic thing, and the purposes of this compound in the preparation antitumor drug.
Remarkable advantage of the present invention: benzoquinones shown in the research is extremely rare by the spirane structure that carbonyl links to each other with oxo bridge with phenyl ring, described benzoquinones spirocyclic compound has significant anti-tumor activity, have not yet to see the report of chemical structure and the cell inhibitory effect activity of this compound, so also there is not yet relevant therewith medicine on the market.
Embodiment
The chemical structure of the compound of indication in following embodiment:
Fermentative production and the separation and purification of embodiment 1 this compound
1 fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, get microorganism Aspergillus aculeatus (
Aspergillus aculeatus) IBPT-3 (has been deposited in Chinese Typical Representative culture collection center on December 24th, 2012, address: Wuhan, China Wuhan University, deposit number is: CCTCC NO:M 2012544) an amount of, and be inoculated on the PDA solid slant culture base and in 28 ℃ of incubators, cultivated 4 days.
Get 4 days microorganism Aspergillus aculeatus of slant culture (
Aspergillus aculeatus) IBPT-3 is an amount of, be inoculated into the 400mL nutrient solution to be housed [nutrient solution forms (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 NaCL 6.0, the water constant volume] the 1000mL Erlenmeyer flask in, 28 ℃ of static cultivations are after 30 days, obtain mycelium and fermented liquid.
The acquisition of 2 medicinal extract
With gauze with mycelium and separation of fermentative broth.With fermented liquid and ethyl acetate 1:2(v/v) extracting twice, the extraction liquid underpressure distillation obtains the ethyl acetate extract of fermented liquid to doing.
The separation and purification of 3 compounds
After medicinal extract is used 100-200 order silica gel mixed sample, take sherwood oil: methylene dichloride: the methyl alcohol gradient composition is as elutriant, by the 300-400 order silica gel silica gel chromatography column chromatography that reduces pressure, collect component Fr.3 (methylene dichloride eluate), Fr.3 is by Sephadex LH-20 gel filtration chromatography, collect the eluate of component Fr.3.2(methylene chloride/methanol v/v=20:1), again take the methylene chloride-methanol gradient composition as elutriant, carry out the pressurized silica gel column chromatography by 300-400 order silica gel, collect the eluate of component Fr.3.2.2(methylene chloride/methanol v/v=50:1), at last by half preparative liquid chromatography (1010 type ODS-A, 10 * 250 mm, 5 μ m): separating flow velocity is 5 mL/min, and moving phase is that 40% acetonitrile contains 0.1% TFA, compound shown in obtaining (6.5 mg, t
R13.4 min).
The compound colorless oil, negative ion HR-ESI-MS
M/z: 345.0981 [M-H]
-, calculated value is 345.0974, molecular formula C
18H
18O
7; UV (MeOH)
λ Max 292 nm; [α]
25 D+ 379.6 ° (
c0.15, MeOH);
1H and
13The NMR data such as C-NMR see Table 1.
Table 1 compound
1H and
13C-NMR data (500 MHz, in DMSO-d
6 )
A)
A) this table signal ownership is based on DEPT, HMQC and HMBC spectrum analysis result.The multiple degree of carbon signal utilizes the DEPT method to determine.
B) numeral in this hurdle and code name represent respectively in HMBC spectrum with corresponding line in
1H provides the coupling coherent signal
13C nuclear.
C) numeral in this hurdle and code name represent respectively in NOE spectrum with corresponding line in
1H provides the coupling coherent signal
1H nuclear.
The test of embodiment 2 anti tumor activity in vitro
1 laboratory sample and experimental technique
The preparation specimen of sample solution is the pure compounds of separation and purification in the above-mentioned enforcement 1.Precision takes by weighing an amount of sample, is mixed with the solution of desired concn with methyl alcohol, and is active for surveying.
The succeeding transfer culture of clone and cell adopts people's lung cancer A549 cell system, leukemia HL-60 cell and K562 cell.Various cells are all with the RPMI-1640 substratum that contains 10%FBS, at 37 ℃ of succeeding transfer culture in the incubator that passes into 5% carbonic acid gas.
The cell inhibitory effect activity test method
People's lung cancer A549 cell that Sulforhodamine B (SRB) method is taken the logarithm vegetative period, being mixed with density with fresh RPMI-1640 substratum is every milliliter 2 * 10
5The cell suspension of individual cell is inoculated in the 96 porocyte culture plates by every hole 200 microlitres, and every hole adds sample or the blank solution of 2 microlitre different concns, in 37 ℃ of lower cultivations 24 hours.Be taken at the cell after cultivating under the drug effect, the morphological change that at first causes in optical microphotograph Microscopic observation drug treating is judged to have or not the cell cycle to suppress the morphological feature of necrocytosis, then 4 ℃, 3000 rev/mins centrifugal 3 minutes, suck supernatant liquor.Add 20% Tricholroacetic Acid, 50 microlitres in every porocyte, place 4 ℃ to fix 1 hour, water flushing 5 times and dry air.Every hole adds acetum 50 microlitres of 0.4% SRB and left standstill 30 minutes in room temperature.Clean 4 times with 1% acetic acid water, remove unconjugated free SRB dyestuff.Every hole adds 150 microlitre Tris and changes (100mmol/L, pH 10.5) soluble protein combination dye into and utilize microplate reader to measure every hole in optical density(OD) (OD) value at 520nm place.Each concentration of sample all arranges three holes in same 96 orifice plates, and other establishes three hole blanks and acellular zeroing hole (if medicine has color will do the acellular zeroing of relative medicine concentration).Each hole OD value is done first corresponding acellular zeroing, gets the average OD value in three holes by IR (%)=(OD again
Blank-OD
Sample)/OD
Blank* 100% formula is calculated the sample on cell proliferation inhibiting rate (IR%) under each concentration.Inhibiting rate according to different concns calculates, and draws IC
50Value.
Leukemia HL-60 cell and K562 cell that tetrazolium (MTT) method is taken the logarithm vegetative period transfer to every milliliter 2 * 10 with cell density
5Individual cell is inoculated in the 96 porocyte culture plates by every hole 200 microlitres, passes into 5% CO in 37 ℃
2Incubator in cultivated 4 hours.Every hole adds 2 microlitre sample liquid or blank solutions, cultivates after 24 hours, and every hole adds MTT liquid (every milliliter of 5 milligrams of normal saline solutions of MTT) 10 microlitres, continue to cultivate 4 hours, 37 ℃, 2000 rev/mins centrifugal 8 minutes, suck supernatant.Every hole adds each 100 microlitre of DMSO, vibrates 15 minutes at micro oscillator, after dissolving fully to crystallization, utilizes MD company to produce SPECTRAMAX Plus type microplate reader and measures every hole in light absorption value (OD) value at 570nm place.Each concentration of sample all arranges three holes in same 96 orifice plates, and other establishes three hole blanks and acellular zeroing hole (if medicine has color will do the acellular zeroing of relative medicine concentration).Each hole OD value is done first corresponding acellular zeroing, gets the average OD value in three holes by IR (%)=(OD again
Blank-OD
Sample)/OD
Blank* 100% formula is calculated cell proliferation inhibition rate under each concentration (IR%).
2. experimental result
Cell inhibitory effect active testing result
In srb assay or mtt assay test, this compound of different concns suppresses to the results are shown in Table 2 to the propagation of people's lung cancer A549 cell, human leukemia HL-60 cell and K562 cell.
Table 2 compound is active to the inhibition of different carcinoma cell proliferation
3. conclusion
Compound has obvious Cytostatic to tumor cell effect, can be used as preparation inhibition of cell proliferation or antineoplastic agent and is used for antineoplastic research.
Claims (3)
1. compound
2. the purposes of compound claimed in claim 1 in preparation cytostatic thing.
3. compound claimed in claim 1 is in the purposes of preparation in the antitumor drug.
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| CN201310016472.1A CN103058971B (en) | 2013-01-17 | 2013-01-17 | Benzoquinone spiro compound derived from aspergillus aculeatus and application of benzoquinone spiro compound |
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| CN201310016472.1A CN103058971B (en) | 2013-01-17 | 2013-01-17 | Benzoquinone spiro compound derived from aspergillus aculeatus and application of benzoquinone spiro compound |
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| CN103058971B CN103058971B (en) | 2014-08-06 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113105428A (en) * | 2021-03-10 | 2021-07-13 | 宁波大学 | Xanthone compound and preparation method and application thereof |
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|---|---|---|---|---|
| CN1139457A (en) * | 1993-12-01 | 1997-01-01 | 诺沃诺尔迪斯克生物技术有限公司 | Aspergillus expression system |
| WO2002074097A1 (en) * | 2001-03-21 | 2002-09-26 | Dsm Ip Assets B.V. | Cheese making process |
| US20030054510A1 (en) * | 1999-08-05 | 2003-03-20 | Soren Ebdrup | Process for the preparation of substituted 3-phenyl-propanoic acid esters and substituted 3-phenyl-propanoic acids |
| WO2009000937A1 (en) * | 2007-06-28 | 2008-12-31 | Dkfz Deutsches Krebsforschungszentrum | Griseofulvin analogues for the treatment of cancer by inhibition of centrosomal clustering |
| CN102277304A (en) * | 2011-08-08 | 2011-12-14 | 南京师范大学 | Aspergillus aculeatus bacterial strain and method for preparing 5,7,8,4'-tetrahydroxyisoflavone by using same |
-
2013
- 2013-01-17 CN CN201310016472.1A patent/CN103058971B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1139457A (en) * | 1993-12-01 | 1997-01-01 | 诺沃诺尔迪斯克生物技术有限公司 | Aspergillus expression system |
| US20030054510A1 (en) * | 1999-08-05 | 2003-03-20 | Soren Ebdrup | Process for the preparation of substituted 3-phenyl-propanoic acid esters and substituted 3-phenyl-propanoic acids |
| WO2002074097A1 (en) * | 2001-03-21 | 2002-09-26 | Dsm Ip Assets B.V. | Cheese making process |
| WO2009000937A1 (en) * | 2007-06-28 | 2008-12-31 | Dkfz Deutsches Krebsforschungszentrum | Griseofulvin analogues for the treatment of cancer by inhibition of centrosomal clustering |
| CN102277304A (en) * | 2011-08-08 | 2011-12-14 | 南京师范大学 | Aspergillus aculeatus bacterial strain and method for preparing 5,7,8,4'-tetrahydroxyisoflavone by using same |
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| Title |
|---|
| MARKUS PAULY，等: "A xyloglucan-specific endo-β-1,4-glucanase from Aspergillus aculeatus:expression cloning in yeast,purification and characterization of the recombinant enzyme", 《GLYCOBIOLOGY》 * |
| 吴少华，等: "印楝内生真菌棘孢曲霉YM311498的代谢产物研究", 《天然产物研究与开发》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113105428A (en) * | 2021-03-10 | 2021-07-13 | 宁波大学 | Xanthone compound and preparation method and application thereof |
| CN113105428B (en) * | 2021-03-10 | 2022-05-17 | 宁波大学 | Xanthone compound and preparation method and application thereof |
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| CN103058971B (en) | 2014-08-06 |
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Address after: 350116, No. 2 School Road, Minhou New District, Fuzhou County, Fuzhou, Fujian Patentee after: Fuzhou University Address before: 350001 No. 523, industrial road, Gulou District, Fujian, Fuzhou Patentee before: Fuzhou University |
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Granted publication date: 20140806 Termination date: 20190117 |