CN104370928A - Indole terpene speradine F derived from aspergillus oryzae and application - Google Patents
Indole terpene speradine F derived from aspergillus oryzae and application Download PDFInfo
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- CN104370928A CN104370928A CN201410202318.8A CN201410202318A CN104370928A CN 104370928 A CN104370928 A CN 104370928A CN 201410202318 A CN201410202318 A CN 201410202318A CN 104370928 A CN104370928 A CN 104370928A
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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 invention relates to an indole terpene speradine F derived from aspergillus oryzae and application. The compound has the effect of inhibiting tumor cell proliferation, and has anti-tumor activity. The structural formula of the compound is shown in the specification. A fermentation product is acquired by performing fermentation culturing on aspergillusoryzae IBPT-1, and then the compound is obtained by performing separation purification on the fermentation product. Experiments confirm that the compound has relatively antitumor activity on human promyelocytic leukemia cell line HL-60, and is applicable to antitumor research by being used to prepare medicines inhibiting cell proliferation or antitumor medicines.
Description
Technical field
The present invention relates to a kind of the indoles terpene speradine F and the application that come from aspergillus oryzae.
Background technology
Indoles terpene is from the secondary metabolite of fungus and bacterium, be separated the compound that the class obtained has unique bioactive, there is the activity suppressing potassium-channel, anti-tumor activity, the activity of selectivity potent PgR competition activity and methicillin-resistant staphylococcus aureus resistance.Cyclopiazonic acid (CPA) Alkaloid is an important branch of indoles terpene compound, and they comprise three structural units usually: an indole ring, a hemiterpene and two acetic acid side chains.Up to now, 6 similar natural product are only had to be found.
The present inventor's research is learnt, aspergillus oryzae (Aspergillus oryzae) IBPT-1, (be deposited in China typical culture collection center on December 1st, 2011, address: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2011437) the crude extract of tunning have good cell inhibitory effect active, then its activeconstituents is studied.Shown in research finds, alkaloid compound has anti-tumor activity, has not yet to see the report of this compound to the proliferation inhibition activity of HL-60 cell, therefore 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 the indoles terpene speradine E and the application that come from aspergillus oryzae.This compound has inhibition tumor cell proliferation function, has anti-tumor activity.
The preparation method of this compound, concrete steps are as follows:
By the ordinary method of culturing micro-organisms, get aspergillus oryzae (
aspergillus oryzae) IBPT-1 is inoculated on PDA solid slant culture base, cultivates 4 days, be then inoculated in nutrient solution in 28 DEG C of incubators, 28 DEG C of static gas wave refrigerator, after 30 days, obtain mycelium and fermented liquid; Described nutrient solution composition: often liter of seawater is containing N.F,USP MANNITOL 20.0g, yeast extract paste 3.0 g, maltose 20.0 g, monosodium glutamate 10.0 g, glucose 10.0 g, KH
2pO
40.5 g, MgSO
40.3 g.
(2) acquisition of medicinal extract
By aspergillus oryzae (
aspergillus oryzae) fermentation culture of IBPT-1, be divided into fermented liquid and mycelium through filtered through gauze.Fermented liquid part by volume 1:2 adds ethyl acetate, after twice extraction, obtains fermented liquid acetic acid ethyl acetate extract.Clear liquid, then with acetone soln ultrasonication 3 times, merges after elimination residue by mycelium, and concentrating under reduced pressure by volume for 1:2 adds extraction into ethyl acetate twice, obtains thalline acetic acid ethyl acetate extract after removing acetone.Fermented liquid acetic acid ethyl acetate extract and thalline acetic acid ethyl acetate extract are merged, underpressure distillation, to dry, obtains the total medicinal extract of extract.
(3) separation and purification of compound
The total medicinal extract of extract is by after 100 ~ 200 order silica gel mixed samples, with sherwood oil: methylene dichloride: methanol gradient component is elutriant, decompression silica gel chromatography column chromatography is carried out by 300-400 order silica gel, be separated into Fr. A, Fr. B, Fr. C, Fr. D, Fr.E is totally 5 components, and wherein Fr. A, Fr. C is two major constituents.Fr. A(sherwood oil: methylene dichloride 1:3 washings) with trichloromethane, methyl alcohol (1:2) is eluent, Sephadex LH-20 gel filtration chromatography is adopted to be separated, obtain Fr. A-1, Fr.A-2 and Fr.A-3 tri-components, Fr. A-2 component (second wash-out component out) carries out pressurized column chromatography by 300-400 order silicagel column again, with methylene dichloride: methyl alcohol is elutriant gradient elution, separation obtains four components: Fr. A-2-1, Fr. A-2-2, Fr. A-2-3 and Fr. A-2-4, wherein, Fr. A-2-1(methylene dichloride washings) by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm): being separated flow velocity is 5 mL/min, moving phase is 60%MeOH, separation obtains compound speradine F 4.2 mg (14.1 min).
The present invention also protects described compound and is preparing the purposes in Cytostatic to tumor cell medicine, and this compound is preparing the purposes in antitumor drug.Tumour cell is HL-60 cell.
Remarkable advantage of the present invention: shown in research, this indoles terpene compound is comparatively rare, described alkaloid compound has significant anti-tumor activity, have not yet to see the report of this compound to HL-60 cell inhibitory effect activity, therefore market also there is not yet medicine related to this.
Accompanying drawing explanation
Fig. 1 is the main COSY of speradine F, HMBC and NOESY signal.
Fig. 2 is the speradine F structure iron obtained by crystalline diffraction.
Embodiment
The chemical structure of the compound of indication in following embodiment:
The fermentative production of this compound of embodiment 1 and separation and purification
1 fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, get aspergillus oryzae (
aspergillus oryzae) IBPT-1 (be deposited in China typical culture collection center, address on December 1st, 2011: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2011437).In right amount, be inoculated on PDA solid slant culture base and cultivate 4 days in 28 DEG C of incubators.
Get the slant culture aspergillus oryzae of 4 days (
aspergillus oryzae) IBPT-1 is appropriate, be inoculated into and 400mL nutrient solution [nutrient solution composition (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 is housed
2pO
40.5, MgSO
40.3, seawater constant volume] 1000mL Erlenmeyer flask in, 28 DEG C of static gas wave refrigerator, after 30 days, obtain mycelium and fermented liquid.
The acquisition of 2 medicinal extract
By aspergillus oryzae (
aspergillus oryzae) fermentation culture of IBPT-1, be divided into fermented liquid and mycelium through filtered through gauze.Fermented liquid part by volume 1:2 adds ethyl acetate, after twice extraction, obtains fermented liquid acetic acid ethyl acetate extract.Clear liquid, then with acetone soln ultrasonication 3 times, merges after elimination residue by mycelium, and concentrating under reduced pressure by volume for 1:2 adds extraction into ethyl acetate twice, obtains thalline acetic acid ethyl acetate extract after removing acetone.Fermented liquid acetic acid ethyl acetate extract and thalline acetic acid ethyl acetate extract are merged, underpressure distillation, to dry, obtains the total medicinal extract of extract.
The separation and purification of 3 compounds
The total medicinal extract of extract is by after 100 ~ 200 order silica gel mixed samples, with sherwood oil: methylene dichloride: methanol gradient component is elutriant, decompression silica gel chromatography column chromatography is carried out by 300-400 order silica gel, be separated into Fr. A, Fr. B, Fr. C, Fr. D, Fr.E is totally 5 components, and wherein Fr. A, Fr. C is two major constituents.Fr. A(sherwood oil: methylene dichloride 1:3 washings) with trichloromethane, methyl alcohol (1:2) is eluent, Sephadex LH-20 gel filtration chromatography is adopted to be separated, obtain Fr. A-1, Fr.A-2 and Fr.A-3 tri-components, Fr. A-2 component (second wash-out component out) carries out pressurized column chromatography by 300-400 order silicagel column again, with methylene dichloride: methyl alcohol is elutriant gradient elution, separation obtains four components: Fr. A-2-1, Fr. A-2-2, Fr. A-2-3 and Fr. A-2-4, wherein, Fr. A-2-1(methylene dichloride washings) by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm): being separated flow velocity is 5 mL/min, moving phase is 60%MeOH, separation obtains compound speradine F 4.2 mg (14.1 min).
Compound as yellow crystals, high resolution mass spectrum HRESI-MS exists
m/z: 415.1506 places provide molecular ion peak [M+H]
+(calcd for C
21h
23n
2o
7, 415.1500), prompting molecular weight is 414, infers that its molecular formula is C in conjunction with spectral information
21h
22n
2o
7.
1h and
13c-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 crystalline diffraction structure iron.
Table 1 NMR compound
1h and
13c-NMR data (500MHz
1h and 125MHz
13c, in CDCl
3)
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 amount of sample, is mixed with the solution of desired concn with methyl alcohol, active for survey.
The succeeding transfer culture of clone and cell adopts tumor cell line, and the DMEM substratum of tumour cell containing 10% FBS, at 37 DEG C of succeeding transfer culture in the incubator passing into 5% carbonic acid gas.
Cell inhibitory effect activity test method (MTT)
The tumour cell of taking the logarithm vegetative period, is adjusted to every milliliter 2 × 10 by cell density
5individual cell, by every hole 200
μl is inoculated in 96 porocyte culture plates, in passing into 5 % CO at 37 DEG C
2incubator in cultivate 4 h.Each sample sets 5 concentration gradients, and each concentration establishes 3 Duplicate Samples, establishes feminine gender, positive control simultaneously, and every hole adds sample liquid or blank solution 2
μl, after cultivating 24 h, every hole adds MTT liquid 10
μl, continue cultivation 4 h, 37 DEG C, centrifugal 8 min of 2000 prm, suck supernatant liquor.Every hole adds DMSO 100
μl, micro oscillator vibrates 15min, dissolves completely to crystallization, measures the light absorption value (OD value) of every hole at 570 nm places by microplate reader.Get three hole mean OD value by formula:
IR (%)=(OD
blank-OD
sample)/OD
blank× 100%
The inhibiting rate (IR) of calculation sample on cell proliferation, and adopt bliss method to calculate half inhibiting rate IC
50and standard variance.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, according to the Cytostatic to tumor cell rate of this compound of different concns, application SPSS16.0 software carries out data processing and calculation of half inhibitory concentration IC
50value.The results are shown in Table 2.
The inhibit activities that table 2 compound is bred human tumor cells
3. conclusion
Compound on tumor cell HL-60 has stronger inhibited proliferation, can be used as preparation HL-60 inhibition of cell proliferation or antineoplastic agent for antineoplastic research.
Claims (5)
1. compound
.
2. the preparation method of compound as claimed in claim 1, it is characterized in that: fermentation culture aspergillus oryzae (Aspergillus oryzae) IBPT-1, obtain fermented product, then from fermented product, separation and purification goes out this compound, described aspergillus oryzae (Aspergillus oryzae) IBPT-1, be deposited in China typical culture collection center, address on December 1st, 2011: Wuhan Wuhan University, deposit number is: CCTCC NO:M 2011437.
3. compound according to claim 1 is preparing the purposes in Cytostatic to tumor cell medicine.
4. compound according to claim 1 is preparing the purposes in antitumor drug.
5. purposes according to claim 3, is characterized in that: described tumour cell is HL-60 cell.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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|---|---|
| CN104370928A true CN104370928A (en) | 2015-02-25 |
| CN104370928B CN104370928B (en) | 2016-06-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110483537A (en) * | 2019-07-22 | 2019-11-22 | 湖南省中医药研究院 | Indoles diterpene-kind compound and its screening technique, separation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2014
- 2014-05-14 CN CN201410202318.8A patent/CN104370928B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|
| CEDRIC W. HOLZAPFEL,等: "Cyclopeamide, an isoindolo[4,6-cd]indole from Penicillium cyclopium", 《PHYTOCHEMISTRY》, vol. 29, no. 2, 15 March 2001 (2001-03-15), pages 639 - 642, XP022515625 * |
| MASASHI TSUDA，等: "Speradine A, a new pentacyclic oxindole alkaloid from a marine-derived fungus Aspergillus tamari", 《TETRAHEDRON》, vol. 59, no. 18, 18 March 2003 (2003-03-18), pages 3227 - 3230, XP004421754, DOI: doi:10.1016/S0040-4020(03)00413-7 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110483537A (en) * | 2019-07-22 | 2019-11-22 | 湖南省中医药研究院 | Indoles diterpene-kind compound and its screening technique, separation method and application |
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