CN104370928B - Come from indole terpene speradine F and the application of aspergillus oryzae - Google Patents
Come from indole terpene speradine F and the application of aspergillus oryzae Download PDFInfo
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- CN104370928B CN104370928B CN201410202318.8A CN201410202318A CN104370928B CN 104370928 B CN104370928 B CN 104370928B CN 201410202318 A CN201410202318 A CN 201410202318A CN 104370928 B CN104370928 B CN 104370928B
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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
- C07D491/22—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 four or more hetero rings
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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
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Abstract
The present invention relates to a kind of indole terpene speradine F 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 separate from fermented product and be purified into this compound.Being verified by experiments, HL-60 HL-60 is had good anti-tumor activity by this compound.Can study for antineoplastic as preparing cytostatic thing or antitumor drug.
Description
Technical field
The present invention relates to a kind of indole terpene speradineF coming from aspergillus oryzae and application.
Background technology
Indole terpene is to separate the class obtained from the secondary metabolite of fungus and antibacterial to have the compound of unique bioactive, there is the activity suppressing potassium-channel, anti-tumor activity, the activity of the potent progesterone receptor competition activity of selectivity and methicillin-resistant staphylococcus aureus resistance.Cyclopiazonic acid (CPA) Alkaloid is an important branch of indole terpene compound, and 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 found.
The present inventor's research is learnt, aspergillus oryzae (Aspergillusoryzae) IBPT-1, (within 1st, it is deposited in China typical culture collection center in December in 2011, address: Wuhan Wuhan University, deposit number is: CCTCCNO:M2011437) the crude extract of tunning have good cell inhibitory effect activity, then its active component is studied.Research finds that shown alkaloid compound has anti-tumor activity, has not yet to see this compound report to the proliferation inhibition activity of HL-60 cell, 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 speradineE coming from aspergillus oryzae and application.This compound has suppression tumor cell proliferation effect, has anti-tumor activity.
The preparation method of this compound, specifically comprises the following steps that
By the conventional method of cultivating microorganism, take aspergillus oryzae (Aspergillusoryzae) IBPT-1 and be inoculated on PDA solid slant culture base, cultivate 4 days in 28 DEG C of incubators, be then seeded in culture fluid, 28 DEG C of static gas wave refrigerator are after 30 days, it is thus achieved that mycelium and fermentation liquid；Described culture fluid forms: every liter of sea water is containing mannitol 20.0g, yeast extract 3.0g, maltose 20.0g, monosodium glutamate 10.0g, glucose 10.0g, KH2PO40.5g、MgSO40.3g。
(2) acquisition of extractum
By the fermentation culture of aspergillus oryzae (Aspergillusoryzae) IBPT-1, it is divided into fermentation liquid and mycelium through filtered through gauze.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 elimination residue by mycelium, after concentrating under reduced pressure removes acetone, by volume for 1:2 addition extraction into ethyl acetate twice, obtains thalline acetic acid ethyl acetate extract.Fermentation liquid acetic acid ethyl acetate extract and thalline acetic acid ethyl acetate extract being merged, decompression is distilled to dry, obtains the total extractum of extract.
(3) separation and purification of compound
The total extractum of extract is by after 100～200 order silica gel mixed samples, with petroleum ether: dichloromethane: methanol gradient component is for eluent, decompression silica gel chromatography column chromatography is carried out by 300-400 order silica gel, it is separated into Fr.A, Fr.B, Fr.C, Fr.D, Fr.E is totally 5 components, and wherein Fr.A, Fr.C are two major constituents.Fr.A(petroleum ether: dichloromethane 1:3 washings) with chloroform, methanol (1:2) is eluant, SephadexLH-20 gel filtration chromatography is adopted to separate, obtain Fr.A-1, three components of Fr.A-2 and Fr.A-3, Fr.A-2 component (second component being eluted out) carries out pressurized column chromatography again by 300-400 order silicagel column, with dichloromethane: methanol is for eluent gradient elution, separation obtains four component: 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 semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250mm, 5 μm): separation flow velocity is 5mL/min, mobile phase is 60%MeOH, separation obtains compound speradineF4.2mg (14.1min).
The present invention also protects described compound purposes in preparing Cytostatic to tumor cell medicine and the purposes that this compound is in preparing antitumor drug.Tumor cell is HL-60 cell.
The remarkable advantage of the present invention: this indole terpene compound shown in research is comparatively rare, described alkaloid compound has significant anti-tumor activity, have not yet to see this compound report to HL-60 cell inhibitory effect activity, therefore market also there is not yet medicine related to this.
Accompanying drawing explanation
Fig. 1 is COSY, HMBC and NOESY signal main for speradineF.
Fig. 2 is the speradineF structure chart obtained by crystal diffraction.
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 (Aspergillusoryzae) IBPT-1 and (within 1st, be deposited in China typical culture collection center in December in 2011, address: Wuhan Wuhan University, deposit number is: CCTCCNO:M2011437).In right amount, it is inoculated on PDA solid slant culture base in 28 DEG C of incubators and cultivates 4 days.
Take the slant culture aspergillus oryzae of 4 days (Aspergillusoryzae) IBPT-1 appropriate, be inoculated into equipped with 400mL culture fluid [culture fluid composition (g/l): 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 (Aspergillusoryzae) IBPT-1, it is divided into fermentation liquid and mycelium through filtered through gauze.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 elimination residue by mycelium, after concentrating under reduced pressure removes acetone, by volume for 1:2 addition extraction into ethyl acetate twice, obtains thalline acetic acid ethyl acetate extract.Fermentation liquid acetic acid ethyl acetate extract and thalline acetic acid ethyl acetate extract being merged, decompression is distilled 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 order silica gel mixed samples, with petroleum ether: dichloromethane: methanol gradient component is for eluent, decompression silica gel chromatography column chromatography is carried out by 300-400 order silica gel, it is separated into Fr.A, Fr.B, Fr.C, Fr.D, Fr.E is totally 5 components, and wherein Fr.A, Fr.C are two major constituents.Fr.A(petroleum ether: dichloromethane 1:3 washings) with chloroform, methanol (1:2) is eluant, SephadexLH-20 gel filtration chromatography is adopted to separate, obtain Fr.A-1, three components of Fr.A-2 and Fr.A-3, Fr.A-2 component (second component being eluted out) carries out pressurized column chromatography again by 300-400 order silicagel column, with dichloromethane: methanol is for eluent gradient elution, separation obtains four component: 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 semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250mm, 5 μm): separation flow velocity is 5mL/min, mobile phase is 60%MeOH, separation obtains compound speradineF4.2mg (14.1min).
Compound as yellow crystals, high resolution mass spectrum HRESI-MS provides molecular ion peak [M+H] at m/z:415.1506 place+(calcdforC21H23N2O7, 415.1500), prompting molecular weight is 414, speculates that its molecular formula is C in conjunction with spectral information21H22N2O7。1H and13C-NMR data are in Table 1, and main COSY, HMBC and NOESY signal is shown in Fig. 1, and Fig. 2 is shown in by crystal diffraction structure chart.
Table 1NMR compound1H and13C-NMR data (500MHz1Hand125MHz13C, inCDCl3)
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 of cell line and cell adopts tumor cell line, and the tumor cell DMEM culture medium containing 10%FBS, at 37 DEG C of successive transfer culture in the incubator passing into 5% carbon dioxide.
Cell inhibitory effect activity test method (MTT)
Take the logarithm the tumor cell of trophophase, cell density is adjusted to every milliliter 2 × 105Individual cell, is inoculated in 96 porocyte culture plates by every hole 200 μ L, in passing into 5%CO at 37 DEG C2Incubator in cultivate 4h.Each sample sets 5 Concentraton gradient, and each concentration sets 3 Duplicate Samples, sets feminine gender, positive control simultaneously, and every hole adds sample liquid or blank solution 2 μ L, and after cultivating 24h, every hole adds MTT liquid 10 μ L, continues to cultivate 4h, 37 DEG C, the centrifugal 8min of 2000prm, sucks supernatant.Every hole adds DMSO100 μ L, and vibrate 15min on micro oscillator, is completely dissolved to crystallization, measures every hole light absorption value (OD value) at 570nm place by microplate reader.Take three hole mean OD value by formula:
IR(%)=(ODBlank-ODSample)/ODBlank×100%
Calculate the suppression ratio (IR) of sample on cell proliferation, and adopt bliss method to calculate half suppression ratio IC50And standard variance.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay is tested, the Cytostatic to tumor cell rate according to this compound of variable concentrations, application SPSS16.0 software carries out data and processes and calculation of half inhibitory concentration IC50Value.Result is in Table 2.
The inhibitory activity that human tumor cells is bred by table 2 compound
3. conclusion
Compound on tumor cell HL-60 has stronger inhibited proliferation, can study for antineoplastic as preparation HL-60 inhibition of cell proliferation or antitumor agent.
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.
4. purposes according to claim 2, it is characterised in that: described tumor cell is HL-60 cell.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410202318.8A CN104370928B (en) | 2014-05-14 | 2014-05-14 | Come from indole terpene speradine F and the application of aspergillus oryzae |
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| CN201410202318.8A CN104370928B (en) | 2014-05-14 | 2014-05-14 | Come from indole terpene speradine F and the application of aspergillus oryzae |
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