CN103058971B - 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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- CN103058971B CN103058971B CN201310016472.1A CN201310016472A CN103058971B CN 103058971 B CN103058971 B CN 103058971B CN 201310016472 A CN201310016472 A CN 201310016472A CN 103058971 B CN103058971 B CN 103058971B
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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 to its secondary metabolite before and had antibacterial, antimycotic, antiviral, desinsection, antitumor isoreactivity preferably, these active compounds can be divided into peptide class by its type of architecture, alkaloids, sesquiterpenoids, fatty acid, xanthone, however the structure that benzoquinones is connected by oxo bridge with phenyl ring rarely has report in the literature.The inventor studies and learns, microorganism Aspergillus aculeatus (
aspergillus aculeatus) IBPT-3 (is 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.Shown in research discovery, benzoquinone compound has anti-tumor activity, has not yet to see the report of chemical structure and the cell inhibitory effect activity of this compound, so on 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 quinone derivatives and application thereof that comes from microorganism Aspergillus aculeatus.This compound has inhibition tumor cell proliferation function, has anti-tumor activity.Its structural formula is:
。
Its constitutional features is: in the molecular skeleton that contains the spirane structure that a benzoquinones is connected with oxo bridge by carbonyl with phenyl ring, molecule four methyl all in contraposition, the unparalleled key of a side in benzoquinones.
The present invention has also protected the purposes of described compound in preparing cytostatic thing, and the purposes of this compound in preparing antitumor drug.
remarkable advantage of the present invention: benzoquinones shown in research is extremely rare with the spirane structure that phenyl ring is connected with oxo bridge by carbonyl, 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 on market, also there is not yet medicine related to this.
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 Wuhan University, deposit number is: CCTCC NO:M 2012544) appropriate, and be inoculated on PDA solid slant culture base and cultivate 4 days in 28 ℃ of incubators.
Get the slant culture microorganism Aspergillus aculeatus of 4 days (
aspergillus aculeatus) IBPT-3 is appropriate, be inoculated into 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, water constant volume] 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 by mycelium and separation of fermentative broth.By fermented liquid and ethyl acetate 1:2(v/v) extracting twice, extraction liquid underpressure distillation, to dry, obtains the ethyl acetate extract of fermented liquid.
The separation and purification of 3 compounds
Medicinal extract is with after 100-200 order silica gel mixed sample, take sherwood oil: methylene dichloride: methyl alcohol gradient composition is 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), take methylene chloride-methanol gradient composition as elutriant again, by 300-400 order silica gel, carry out pressurized silica gel column chromatography, collect the eluate of component Fr.3.2.2(methylene chloride/methanol v/v=50:1), finally by half preparative liquid chromatography (1010 type ODS-A, 10 * 250 mm, 5 μ m): separated flow velocity is 5 mL/min, moving phase is that 40% acetonitrile is containing 0.1% TFA, (6.5 mg of compound shown in obtaining, t
r13.4 min).
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 are in 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 DEPT method to determine.
B) numeral in this hurdle and code name represent respectively in HMBC spectrum with corresponding line in
1h provides coupling coherent signal
13c core.
C) numeral in this hurdle and code name represent respectively in NOE spectrum with corresponding line in
1h provides coupling coherent signal
1h core.
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 sample, is mixed with the solution of desired concn with methyl alcohol, 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 containing 10%FBS, at 37 ℃ of succeeding transfer culture in the incubator that passes into 5% carbonic acid gas.
Cell inhibitory effect activity test method
People's lung cancer A549 cell that Sulforhodamine B (SRB) method is taken the logarithm vegetative period, with fresh RPMI-1640 substratum, being mixed with density is every milliliter 2 * 10
5the cell suspension of individual cell, is inoculated in 96 porocyte culture plates by every hole 200 microlitres, and every hole adds sample or the blank solution of 2 microlitre different concns, cultivates 24 hours at 37 ℃.Be taken at the cell after cultivation under drug effect, first the morphological change causing in optical microphotograph Microscopic observation drug treating, judgement has or not the cell cycle to suppress, the morphological feature of necrocytosis, then 4 ℃, 3000 revs/min centrifugal 3 minutes, suck supernatant liquor.In every porocyte, add 20% Tricholroacetic Acid 50 microlitres, be placed in 4 ℃ and fix 1 hour, water rinses 5 times and dry air.Every hole adds acetum 50 microlitres of 0.4% SRB and in room temperature standing 30 minutes.With 1% acetic acid water, clean 4 times, remove unconjugated free SRB dyestuff.Every hole adds 150 microlitre Tris to change (100mmol/L, pH 10.5) soluble protein combination dye into and utilizes microplate reader to measure every hole in optical density(OD) (OD) value at 520nm place.In same 96 orifice plates, each concentration of sample all arranges three holes, separately 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 first done corresponding acellular zeroing, then gets the three average OD values in hole by IR (%)=(OD
blank-OD
sample)/OD
blank* 100% formula is calculated the sample on cell proliferation inhibiting rate (IR%) under each concentration.According to the inhibiting rate of different concns, calculate, draw IC
50value.
Leukemia HL-60 cell and K562 cell that tetrazolium (MTT) method is taken the logarithm vegetative period, be adjusted to every milliliter 2 * 10 by cell density
5individual cell, is inoculated in 96 porocyte culture plates by every hole 200 microlitres, in 37 ℃, passes into 5% CO
2incubator in cultivate 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 revs/min centrifugal 8 minutes, suck supernatant.Every hole adds each 100 microlitres of DMSO, vibrates 15 minutes on micro oscillator, after dissolving completely, utilizes MD company to produce SPECTRAMAX Plus type microplate reader and measures every hole in light absorption value (OD) value at 570nm place to crystallization.In same 96 orifice plates, each concentration of sample all arranges three holes, separately 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 first done corresponding acellular zeroing, then gets the three average OD values in hole by IR (%)=(OD
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 and prepares inhibition of cell proliferation or antineoplastic agent for antineoplastic research.
Claims (3)
1. compound
.
2. the purposes of compound claimed in claim 1 in preparing cytostatic thing.
3. the purposes of compound claimed in claim 1 in preparing antitumor drug.
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Citations (5)
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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 |
Non-Patent Citations (2)
| Title |
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| 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》.1999,第9卷(第1期),第93-100页. * |
| 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》.2009,第21卷第731-732页. * |
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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 |