CN107739361B - Derived from aspergillus versicolor anthraquinone analog compound and prepare the application of anti-human colon cancer drug - Google Patents
Derived from aspergillus versicolor anthraquinone analog compound and prepare the application of anti-human colon cancer drug Download PDFInfo
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- CN107739361B CN107739361B CN201711079824.2A CN201711079824A CN107739361B CN 107739361 B CN107739361 B CN 107739361B CN 201711079824 A CN201711079824 A CN 201711079824A CN 107739361 B CN107739361 B CN 107739361B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
- C07D313/16—Eight-membered rings
- C07D313/20—Eight-membered rings condensed with carbocyclic rings or ring systems
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- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
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Abstract
The present invention relates to be derived from aspergillus versicolor anthraquinone analog compound and prepare the application of anti-human colon cancer drug.The compound, which has, inhibits Colon Cancer Cells effect.Its structural formula are as follows:.By fermented and cultured aspergillus versicolor (Aspergillus versicolor) IBPT-7, fermentation material is obtained, the compound is then isolated and purified out from fermentation material.It is verified by experiments, which has preferable anti-tumor activity to human colon cancer cell HCT-116.It can be used as the research for preparing that Proliferation of Human Colon inhibits drug or anti-tumor drug is used for human colon carcinoma.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to one kind is derived from aspergillus versicolor anthraquinone analog compound and prepares anti-human
The application of colon cancer drug.
Background technique
Anthraquinone analog compound exists extensively in natural drug and has important pharmacological effect, such as Chinese medicine rhizoma polygonati, Asian puccoon, pellet
All contain a large amount of anthraquinone analog compounds in the plants such as ginseng, rheum officinale, tripterygium wilfordii, there are a variety of function such as antitumor, antibacterial, anti-oxidant
Effect is especially widely used in the prevention and treatment of the major diseases such as cancer, cardiovascular disease, senile dementia, AIDS, and research achievement is many
More, by global scientist and medical personage common concerns.Wherein in antitumor field, the mechanism of action, which is mainly manifested in, to be lured
Lead apoptosis of tumor cells, the effect of reversing tumor cell multidrug resistance, enzyme inhibitor, the proliferation and cell week for influencing tumour cell
Phase, works to killing and mutagenesis of tumour cell etc. at the metabolism for inhibiting tumour cell.Research in recent years shows that some oceans are true
Bacterium can also generate structure novel, activity good anthraquinone analog compound during cometabolism, have medicinal well and produce
Industry prospect.
The present inventor's research learns that aspergillus versicolor (has been deposited in Chinese Typical Representative culture guarantor on December 25th, 2013
Hiding center, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013715) tunning coarse extraction
Object has good cell inhibitory effect activity, studies then its active constituent.Research finds shown anthraquinone analog compound tool
There is anti-human colon cancer reactive, has not yet to see the compound to the report of the proliferation inhibition activity of human colon cancer cell, therefore city
Also there is not yet drug related to this on field.
Summary of the invention
The purpose of the present invention is to provide one kind derived from aspergillus versicolor anthraquinone analog compound and to prepare anti-human colon cancer drug
Application.The compound, which has, inhibits Proliferation of Human Colon effect, has anti-human colon cancer reactive.Its structural formula are as follows:
。
The preparation method of the compound is to obtain fermentation material by fermented and cultured aspergillus versicolor, then divides from fermentation material
From being purified into the compound.Specific step is as follows:
1 fermenting and producing
The conventional method for cultivating microorganism, takes aspergillus versicolor to be inoculated into PDA solid slope culture medium in 28 DEG C of incubators
Middle culture 2 to 3 days, is then seeded into SWS culture solution, and 28 DEG C after static gas wave refrigerator 30 days, obtain mycelium and 20 liters of fermentation liquid；
Culture solution composition: starch-containing 10.0 g of every liter of water, 1.0 20.0 g of g, NaCl of peptone.
The acquisition of 2 medicinal extract
By aspergillus versicolor fermentation culture (about 20 L), via being divided into mycelium and fermentation liquid two parts after filtered through gauze.
Ethyl acetate is added in 1:2 by volume for fermentation liquid part, is extracted twice, and it is dense that resulting acetic acid ethyl acetate extract is merged decompression
It is reduced to and closely does, collect fermentation liquid ethyl acetate extract.Mycelium then with the aqueous solution ultrasonication of the acetone containing 70%-80% 3 times, removes
Acetone is removed through being concentrated under reduced pressure after going residue to merge clear liquid, ethyl acetate is added for 1:2 by volume and is extracted twice, depressurizes dense
It is reduced to and closely does to obtain mycelium medicinal extract.Extract leaching is obtained after fermentation liquid ethyl acetate extract and mycelium medicinal extract two parts are merged
Cream totally 6.0 g.
The separation and purification of 3 compounds
Medicinal extract (6.0 g) is by 100-200 mesh silica gel mixed sample, using petroleum ether: methylene chloride: methanol as gradient eluent,
Carry out decompression silica gel chromatographic column chromatography.By simple thin-layer chromatographic analysis, merges, be separated into component A-F.Component E (1.1 g)
(methylene chloride: the eluate of methanol v/v=100:1) is using methylene chloride: methanol carries out pressured column silica gel color as gradient elution agent
Spectrum chromatography, merges after thin-layer chromatographic analysis and obtains five subfraction E1-E5.Component E2 (0.3 g) is with chloroform: first
Alcohol=1:2 is eluant, eluent, is carried out gel filtration chromatography (Sephadex LH-20), merges after thin-layer chromatographic analysis and obtains four
Subfraction E2-1 ~ E2-4.The subfraction E2-2 (93 mg) of E2 by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250
Mm, 5 μm): separation flow velocity is 5 mL/min, and mobile phase is that 75% acetonitrile contains 0.1% TFA, obtain shown compound (3.4 mg,
tR 9.1 min)。
The aspergillus versicolor (Aspergillus versicolor) IBPT-7, it is deposited in on December 25th, 2013
China typical culture collection center, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013715.
The present invention also protects the compound to inhibit the application in Proliferation of Human Colon drug in preparation, and should
Compound is preparing the application in anti-human colon cancer drug.
Remarkable advantage of the invention: the anthraquinone compounds shown in studying have not been reported and have significant inhibition human colon carcinoma
Proliferation activity, has not yet to see the compound to the report of Proliferation of Human Colon inhibitory activity, in the market also there is not yet
Drug related to this.
Detailed description of the invention
The main COSY of Fig. 1 compound, HMBC and NOE signal.
Specific embodiment
The chemical structure of signified compound in the following example:
The fermenting and producing and separation and purification of 1 compound of embodiment
1 fermenting and producing
Produce bacterium fermented and cultured: by culture microorganism conventional method, take aspergillus versicolor (Aspergillus versicolor) IBPT-7 (is deposited in China typical culture collection center on December 25th, 2013, address: Wuhan
Wuhan University, deposit number are: CCTCC NO:M 2013715) in right amount, it is inoculated into PDA solid slope culture medium at 28 DEG C
It is cultivated 2 to 3 days in incubator.
It takes inclined-plane culture 2 to 3 days aspergillus versicolors appropriate, is inoculated into SWS culture solution, 28 DEG C after static gas wave refrigerator 30 days,
Obtain mycelium and fermentation liquid 20L；The culture solution composition: starch-containing 10.0 g of every liter of water, 1.0 g, NaCl 20.0 of peptone
g。
The acquisition of 2 medicinal extract
By aspergillus versicolor fermentation culture (about 20 L), via being divided into mycelium and fermentation liquid two parts after filtered through gauze.
Ethyl acetate is added in 1:2 by volume for fermentation liquid part, is extracted twice, and it is dense that resulting acetic acid ethyl acetate extract is merged decompression
It is reduced to and closely does, collect fermentation liquid ethyl acetate extract.Mycelium then with the aqueous solution ultrasonication of the acetone containing 70%-80% 3 times, removes
Acetone is removed through being concentrated under reduced pressure after going residue to merge clear liquid, ethyl acetate is added for 1:2 by volume and is extracted twice, depressurizes dense
It is reduced to and closely does to obtain mycelium medicinal extract.Extract leaching is obtained after fermentation liquid ethyl acetate extract and mycelium medicinal extract two parts are merged
Cream totally 6.0 g.
The separation and purification of 3 compounds
Medicinal extract (6.0 g) is by 100-200 mesh silica gel mixed sample, using petroleum ether: methylene chloride: methanol as gradient eluent,
Carry out decompression silica gel chromatographic column chromatography.By simple thin-layer chromatographic analysis, merges, be separated into component A-F.Component E (1.1 g)
(methylene chloride: the eluate of methanol v/v=100:1) is using methylene chloride: methanol carries out pressured column silica gel color as gradient elution agent
Spectrum chromatography, merges after thin-layer chromatographic analysis and obtains five subfraction E1-E5.Component E2 (0.3 g) is with chloroform: first
Alcohol=1:2 is eluant, eluent, is carried out gel filtration chromatography (Sephadex LH-20), merges after thin-layer chromatographic analysis and obtains four
Subfraction E2-1 ~ E2-4.The subfraction E2-2 (93 mg) of E2 by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250
Mm, 5 μm): separation flow velocity is 5 mL/min, and mobile phase is that 75% acetonitrile contains 0.1% TFA, obtain shown compound (3.4 mg,
tR 9.1 min)。
It is yellow powder under compound room temperature, high-resolution electrospray ionization mass spectrum HRESI-MS existsm/z: 383.1161 [M-H]
ˉ, calcd for C21H19O7, 383.1131, prompting molecular weight is 384, speculates that molecular formula is in conjunction with spectral information
C21H20O7。1H and13C-NMR data are shown in Table 1, and main COSY, HMBC and NOE signal is shown in Fig. 1.
1 compound of table1H and13C-NMR data (500 MHz1H and 126 MHz 13C, in DMSO-d 6 )
The test of 2 anti tumor activity in vitro of embodiment
1 laboratory sample and experimental method
The preparation test sample of sample solution is the pure compounds of separation and purification in above-described embodiment 1.Precision claims
Appropriate amount of sample is taken, the solution of required concentration is configured to methanol, for surveying activity.
The squamous subculture of cell line and cell uses tumor cell line, and tumour cell uses the DMEM containing 10% FBS to cultivate
Base, at 37 DEG C in being passed through 5% CO2Incubator in squamous subculture.
Cell inhibitory effect activity test method
The tumour cell of tetrazolium (MTT) method logarithmic growth phase, is adjusted to every milliliter 1 × 10 for cell density5It is a thin
Born of the same parents are inoculated in 96 porocyte culture plates by every 200 μ L of hole, are passed through 5% CO in 37 DEG C2Incubator in cultivate 4h.Every hole adds
Enter the sample liquid or blank solution of 2 μ L, after culture for 24 hours, MTT liquid (every mL5mg normal saline solution of MTT) 10 is added in every hole
μ L continues to cultivate 4h, and 37 DEG C, 2000 turns/min centrifugation 8min draw supernatant.Each 100 μ L of DMSO is added in every hole, in micro oscillation
15min is vibrated on device, until measuring after crystallization is completely dissolved using the SPECTRAMAX Plus type microplate reader of MD company production
Extinction (OD) value of every hole at 570 nm.Each concentration of sample is respectively provided with three holes in 96 orifice plate of same, is separately arranged three
The blank control in hole and cell-free withered hole (if drug there is color to do relative medicine concentration cell-free withered).Each hole OD value
It first does corresponding cell-free withered, then takes three hole mean OD values by IR (%)=(ODBlank control-ODSample)/ODBlank control× 100% calculates
The proliferation inhibition rate (IR%) of cell under each concentration.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, according to the Cytostatic to tumor cell rate of the compound of various concentration, using SPSS16.0
Software carries out data processing and calculation of half inhibitory concentration IC50Value.It the results are shown in Table 2.
Inhibitory activity of 2 compound of table to Proliferation of Human Colon
3. conclusion
The compound has preferable anti-tumor activity to human colon cancer cell.It can be used as and prepare Proliferation of Human Colon
Drug or anti-tumor drug is inhibited to be used for the research of human colon carcinoma.
Claims (2)
1. compoundInhibit the application in Proliferation of Human Colon drug in preparation.
2. compoundPreparing the application in anti-human colon cancer drug.
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| CN108753627B (en) * | 2018-06-15 | 2022-04-22 | 河北大学 | Marine aspergillus derived oxaanthraquinone compound, preparation method thereof and application thereof in preparation of antitumor agent |
| CN115232096B (en) * | 2022-06-15 | 2023-05-05 | 福建卫生职业技术学院 | Lactone compound from aspergillus flavus, preparation method thereof and application thereof in anti-human liver cancer drugs |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104478891A (en) * | 2014-12-18 | 2015-04-01 | 福州大学 | Penicillium citrinum sourced citrinin compound (penicitrinol O) as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104478891A (en) * | 2014-12-18 | 2015-04-01 | 福州大学 | Penicillium citrinum sourced citrinin compound (penicitrinol O) as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| １株海洋来源真菌Ｎｉｇｒｏｓｐｏｒａｓｐ．蒽醌类化合物及其生物活性研究;邢倩等;《中国海洋药物》;20141231;8-14 * |
| 海洋生物内生真菌中蒽醌类次生代谢产物的研究;霍娟等;《中国海洋药物杂志》;20100630;48-54 * |
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