CN104387396B - Come from indole terpene speradine E and the application of aspergillus oryzae - Google Patents
Come from indole terpene speradine E and the application of aspergillus oryzae Download PDFInfo
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- CN104387396B CN104387396B CN201410201856.5A CN201410201856A CN104387396B CN 104387396 B CN104387396 B CN 104387396B CN 201410201856 A CN201410201856 A CN 201410201856A CN 104387396 B CN104387396 B CN 104387396B
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- aspergillus oryzae
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- indole
- speradine
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- 0 C*(c1cccc2cc(C(C)(C)*(C(CCO)=O)C3=*)c3c3c12)*3=O Chemical compound C*(c1cccc2cc(C(C)(C)*(C(CCO)=O)C3=*)c3c3c12)*3=O 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/06—Peri-condensed systems
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- 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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Abstract
The present invention relates to a kind of indole terpene speradine E coming from aspergillus oryzae and application.This compound has suppression tumor cell proliferation effect, has anti-tumor activity.Its structural formula is:.By fermentation culture aspergillus oryzae (Aspergillus oryzae) IBPT 1, obtain fermented product, then from fermented product isolated and purified go out this compound.Being verified by experiments, this compound has preferable anti-tumor activity to Human cervical cancer cell lines hela.Can study for antineoplastic as preparing cytostatic thing or antitumor drug.
Description
Technical field
The present invention relates to a kind of indole terpene speradine E coming from aspergillus oryzae and application.
Background technology
Indole terpene is that a class of isolated from the secondary metabolite of fungus and antibacterial has unique bioactive
Compound, has the activity of suppression potassium-channel, anti-tumor activity, selectivity potent progesterone receptor competition activity and anti-resistance to first
The activity of oxygen XiLin staphylococcus aureus.Cyclopiazonic acid (CPA) Alkaloid is a weight of indole terpene compound
Wanting branch, they generally comprise three construction units: an indole ring, a hemiterpene and two acetic acid side chains.Up to now, only
6 similar natural prodcuts are had to be found.
The present inventor's research is learnt, aspergillus oryzae (Aspergillus oryzae) IBPT-1, (in December in 2011 1 day
Being deposited in China typical culture collection center, address: Wuhan Wuhan University, deposit number is: CCTCC NO:M
2011437) crude extract of tunning has good cell inhibitory effect activity, then studies its active component.
Research finds that shown alkaloid compound has anti-tumor activity, has not yet to see this compound and presses down the propagation of hela cell
The report of system activity, therefore also there is not yet medicine related to this on market.
Summary of the invention
It is an object of the invention to provide a kind of indole terpene speradine E coming from aspergillus oryzae and application.This compound
There is suppression tumor cell proliferation effect, there is anti-tumor activity.Its structural formula is:
。
The preparation method of this compound, specifically comprises the following steps that
By the conventional method of cultivating microorganism, take aspergillus oryzae (Aspergillus oryzae) IBPT-1 and be inoculated into PDA admittedly
On body slant medium, cultivating 4 days, be then seeded in culture fluid in 28 DEG C of incubators, 28 DEG C of static gas wave refrigerator, after 30 days, obtain
Obtain mycelium and fermentation liquid;Described culture fluid forms: every liter of sea water is containing mannitol 20.0g, yeast extract 3.0 g, maltose 20.0
G, monosodium glutamate 10.0 g, glucose 10.0 g, KH2PO4 0.5 g、MgSO4 0.3 g。
(2) acquisition of extractum
By the fermentation culture of aspergillus oryzae (Aspergillus oryzae) IBPT-1, through filtered through gauze be divided into fermentation liquid and
Mycelium.Fermentation liquid part 1:2 by volume adds ethyl acetate, after twice extraction, obtains fermentation liquid acetic acid ethyl acetate extract.
Clear liquid, then with acetone soln ultrasonication 3 times, is merged after filtering off residue, after concentrating under reduced pressure removes acetone, by volume by mycelium
It is extracted twice than adding ethyl acetate for 1:2, obtains thalline acetic acid ethyl acetate extract.By fermentation liquid acetic acid ethyl acetate extract and thalline
Acetic acid ethyl acetate extract merges, and decompression distillation, to dry, obtains the total extractum of extract.
(3) separation and purification of compound
The total extractum of extract is by after 100~200 mesh silica gel mixed samples, with petroleum ether: dichloromethane: methanol gradient component is
Eluent, carries out, by 300-400 mesh silica gel, the silica gel chromatography column chromatography that reduces pressure, is separated into Fr. A, Fr. B, Fr. C, Fr.
D, Fr.E totally 5 components, wherein Fr. A, Fr. C is two major constituents.Fr. A(petroleum ether: dichloromethane 1:3 washings)
With chloroform, methanol (1:2) as eluant, use Sephadex LH-20 gel filtration chromatography separate, obtain Fr. A-1,
Tri-components of Fr.A-2 and Fr.A-3, Fr. A-2 component (second component being eluted out) is again by 300-400 mesh silica gel
Post carries out pressurized column chromatography, with dichloromethane: methanol is eluent gradient elution, four components of isolated: Fr. A-2-1,
Fr. A-2-2, Fr. A-2-3 and Fr. A-2-4, wherein, Fr. A-2-1(dichloromethane washings) by half preparation liquid phase color
Spectrum (1010 types ODS-A, 10 × 250 mm, 5 μm): separating flow velocity is 5 mL/min, and flowing is 60%MeOH mutually, isolated
Compound speradine E 3.2 mg (17.3 min).
Described aspergillus oryzae (Aspergillus oryzae) IBPT-1, is deposited in Chinese Typical Representative on 1st in December in 2011
Culture collection center, address: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2011437.
The present invention also protects described compound purposes in preparing Cytostatic to tumor cell medicine, and this chemical combination
Thing purposes in preparing antitumor drug.Described tumor cell is hela cell.
The remarkable advantage of the present invention: this indole terpene compound shown in research is the rarest, described alkaloid compound has
There is significant anti-tumor activity, have not yet to see this compound report to hela cell inhibitory effect activity, therefore on market
Also there is not yet medicine related to this.
Accompanying drawing explanation
Fig. 1 is the COSY that speradine E is main, HMBC signal.
Detailed description of the invention
The chemical constitution of the compound of indication in examples below:
The fermenting and producing of this compound of embodiment 1 and separation and purification
1 fermenting and producing
Produce the fermentation culture of bacterium: by the conventional method of cultivating microorganism, take aspergillus oryzae (Aspergillus oryzae)
IBPT-1 (is deposited in China typical culture collection center on 1st in December in 2011, address: Wuhan Wuhan University, preservation
Numbering is: CCTCC NO:M 2011437).In right amount, it is inoculated on PDA solid slant culture base in 28 DEG C of incubators, cultivates 4
My god.
Take the slant culture aspergillus oryzae of 4 days (Aspergillus oryzae) IBPT-1 appropriate, be inoculated into and train equipped with 400mL
Nutrient solution [culture fluid composition (grams per liter): mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0,
KH2PO40.5, MgSO40.3, sea water constant volume] 1000mL conical flask in, 28 DEG C of static gas wave refrigerator are after 30 days, it is thus achieved that mycelium
And fermentation liquid.
The acquisition of 2 extractum
By the fermentation culture of aspergillus oryzae (Aspergillus oryzae) IBPT-1, through filtered through gauze be divided into fermentation liquid and
Mycelium.Fermentation liquid part 1:2 by volume adds ethyl acetate, after twice extraction, obtains fermentation liquid acetic acid ethyl acetate extract.
Clear liquid, then with acetone soln ultrasonication 3 times, is merged after filtering off residue, after concentrating under reduced pressure removes acetone, by volume by mycelium
It is extracted twice than adding ethyl acetate for 1:2, obtains thalline acetic acid ethyl acetate extract.By fermentation liquid acetic acid ethyl acetate extract and thalline
Acetic acid ethyl acetate extract merges, and decompression distillation, to dry, obtains the total extractum of extract.
The separation and purification of 3 compounds
The total extractum of extract is by after 100~200 mesh silica gel mixed samples, with petroleum ether: dichloromethane: methanol gradient component is
Eluent, carries out, by 300-400 mesh silica gel, the silica gel chromatography column chromatography that reduces pressure, is separated into Fr. A, Fr. B, Fr. C, Fr.
D, Fr.E totally 5 components, wherein Fr. A, Fr. C is two major constituents.Fr. A(petroleum ether: dichloromethane 1:3 washings)
With chloroform, methanol (1:2) as eluant, use Sephadex LH-20 gel filtration chromatography separate, obtain Fr. A-1,
Tri-components of Fr.A-2 and Fr.A-3, Fr. A-2 component (second component being eluted out) is again by 300-400 mesh silica gel
Post carries out pressurized column chromatography, with dichloromethane: methanol is eluent gradient elution, four components of isolated: Fr. A-2-1,
Fr. A-2-2, Fr. A-2-3 and Fr. A-2-4, wherein, Fr. A-2-1(dichloromethane washings) by half preparation liquid phase color
Spectrum (1010 types ODS-A, 10 × 250 mm, 5 μm): separating flow velocity is 5 mL/min, and flowing is 60%MeOH mutually, isolated
Compound speradine E 3.2 mg (17.3 min).
The unformed powder of compound orange, high resolution mass spectrum HRESI-MS provides molecular ion at m/z:389.1108
Peak [M+Na]+, (Calcd. for C20H18N2NaO5, 389.1113), prompting molecular weight is 366, speculates in conjunction with spectral information
Its molecular formula is C20H18N2O5。1H and13C-NMR data are shown in Table 1, and main COSY and HMBC is shown in Fig. 1.
Table 1 NMR compound1H and13C-NMR data (500MHz1H and 125MHz 13C, in CDCl3)a)
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 weighs
Appropriate amount of sample, is configured to the solution of desired concn with methanol, for surveying activity.
The successive transfer culture employing tumor cell line of cell line and cell, the tumor cell DMEM culture medium containing 10% FBS,
At 37 DEG C of successive transfer culture in the incubator being passed through 5% carbon dioxide.
Cell inhibitory effect activity test method
Sulforhodamine B (SRB) method is taken the logarithm the hela cell of trophophase, cultivates basigamy with fresh RPMI-1640
Making density is every milliliter 2 × 105The cell suspension of individual cell, is inoculated in 96 porocyte culture plates by every hole 200 microlitre, often
Hole adds sample or the blank solution of 2 microlitre variable concentrations, cultivates 24 hours at 37 DEG C.After being taken under medicine effect cultivation
Cell, observes the morphological change that drug treating causes, it is judged that the most under an optical microscope with or without Cyclin-dependent kinase, cell
Downright bad morphological feature, is then centrifuged 3 minutes at 4 DEG C, 3000 revs/min, sucks supernatant.Every porocyte adds 20%
Trichloroacetic acid 50 microlitre, is placed in 4 DEG C and fixes 1 hour, rinses 5 times with water and air is dried.Every hole adds the acetic acid of 0.4% SRB
Solution 50 microlitre also stands 30 minutes in room temperature.Clean 4 times with 1% acetic acid water, remove unconjugated free SRB dyestuff.Every hole adds
Enter 150 microlitre Tris change (100mmol/L, pH 10.5) soluble protein combination dye into and utilize the every hole of microplate reader mensuration to exist
Optical density (OD) value at 520nm.In same 96 orifice plate, each concentration of sample is respectively provided with three holes, separately sets three hole blank right
According to acellular zeroing hole (if medicine has color relative medicine the to be done acellular zeroing of concentration).Each hole OD value first does corresponding nothing
Cell returns to zero, then takes three hole mean OD value by IR (%)=(OD blank-OD sample)/OD blank × 100% formula
Calculate sample cell proliferation suppression ratio (IR%) under each concentration.Suppression ratio according to variable concentrations calculates, and draws IC50
Value.
2. experimental result
Cell inhibitory effect active testing result
In srb assay is tested, according to the Cytostatic to tumor cell rate of this compound of variable concentrations, apply SPSS16.0
Software carries out data and processes and calculation of half inhibitory concentration IC50Value.The results are shown in Table 2.
The inhibitory activity that human tumor cells is bred by table 2 compound
3. conclusion
Compound on tumor cell hela has stronger inhibited proliferation, can be as preparation hela cell inhibitory effect
Agent or antitumor agent are studied for antineoplastic.
Claims (4)
1. compound。
2. the purposes in preparing Cytostatic to tumor cell medicine of the compound described in claim 1.
3. the purposes in preparing antitumor drug of the compound described in claim 1.
Purposes the most according to claim 2, it is characterised in that: described tumor cell is hela cell.
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CN107496420B (en) * | 2017-08-25 | 2020-07-07 | 中国科学院微生物研究所 | Application of cyclopiazonic acid alkaloid compound |
CN114409660B (en) * | 2022-01-27 | 2023-03-17 | 云南中烟工业有限责任公司 | CPA type indole alkaloid compound and preparation method and application thereof |
CN114409661B (en) * | 2022-01-27 | 2023-02-28 | 云南中烟工业有限责任公司 | Indole alkaloid compound and preparation method and application thereof |
Citations (1)
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CN103183666A (en) * | 2011-12-28 | 2013-07-03 | 中国科学院沈阳应用生态研究所 | Cyclopiazonic acid compound, and preparation and application thereof |
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CN103183666A (en) * | 2011-12-28 | 2013-07-03 | 中国科学院沈阳应用生态研究所 | Cyclopiazonic acid compound, and preparation and application thereof |
Non-Patent Citations (2)
Title |
---|
Cyclopeamide, an isoindolo[4,6-cd]indole from Penicillium cyclopium;CEDRIC W. HOLZAPFEL,等;《Phytochemistry》;20010315;第29卷(第2期);全文 * |
Speradine A, a new pentacyclic oxindole alkaloid from a marine-derived fungus Aspergillus tamari;Masashi Tsuda,等;《Tetrahedron》;20030318;第59卷(第18期);全文 * |
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