CN107298669A - Come from the secalonic acid I of penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application - Google Patents
Come from the secalonic acid I of penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application Download PDFInfo
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- CN107298669A CN107298669A CN201710460060.5A CN201710460060A CN107298669A CN 107298669 A CN107298669 A CN 107298669A CN 201710460060 A CN201710460060 A CN 201710460060A CN 107298669 A CN107298669 A CN 107298669A
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- penicillium oxalicum
- epidermoid carcinoma
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- secalonic acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
- C07D311/84—Xanthenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 9
- C07D311/86—Oxygen atoms, e.g. xanthones
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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
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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Abstract
The present invention relates to the secalonic acid I for coming from penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application.The compound, which has, suppresses human mouth epidermoid carcinoma cel l proliferation.Its structural formula is：.By fermented and cultured penicillium oxalicum (Penicillium oxalicum) IBPT 6, fermentate is obtained, the compound is then isolated and purified out from fermentate.It is verified by experiments, the compound has preferable antitumor activity to human mouth epidermoid carcinoma cell KB.The research of human mouth epidermoid carcinoma can be used for as human mouth epidermoid carcinoma cytostatic thing or antineoplastic is prepared.
Description
Technical field
The present invention relates to the secalonic acid I for coming from penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application, belong to medicine
Biological field.
Background technology
Secalonic acid class compound（Secalonic acids）Belong to ergot pigment（Ergochrome）Secondary metabolism is produced
Thing, is xanthone dimer.Since Stoll etc. was in the isolated secalonic acid A from fungi in 1952
（Secalonic acid A）Afterwards, the serial compound secalonic acid（A, B, C, D, E, F, G）Just constantly by
It was found that and research.Secalonic acid class compound has a variety of physiologically actives, with secalonic acid D（Secalonic acid
D, SAD）Exemplified by, 5 mg/ml SAD is added in physiological saline, in the range of 5-20 mg, you can to treat early stage bladder
In the range of cancer, 50-100 mg, and be free from side effects generation effective in cure to more serious carcinoma of urinary bladder.It has been investigated that, some seas
Foreign fungi can produce the secalonic acid class compound that structure is novel, activity is good during cometabolism, with good medicine
With and industrialization prospect.
The present inventor studies and learnt, penicillium oxalicum (Penicillium oxalicum) IBPT-6, (in 2013
December 25 was deposited in China typical culture collection center, address:Wuhan Wuhan University, deposit number is：CCTCC NO：
M 2013714) the crude extract of tunning have good cell inhibitory effect activity, its active component is ground then
Study carefully.Research finds that shown secalonic acid class compound has anti-human oral cavity epidermoid carcinoma activity, has not yet to see the compound pair
The report of the proliferation inhibition activity of human mouth epidermoid carcinoma cell, therefore in the market is also there is not yet medicine related to this.
The content of the invention
It is an object of the invention to provide a kind of secalonic acid class compound Secalonic acid for coming from penicillium oxalicum
Application in terms of I preparation methods and its suppression human mouth epiderm-like cancer cell multiplication.The compound, which has, suppresses oral cavity epiderm-like
Cancer cell multiplication is acted on, with anti-human oral cavity epidermoid carcinoma activity.Its structural formula is：
。
The preparation method of the compound, be by fermented and cultured penicillium oxalicum (Penicillium oxalicum)
IBPT-6, obtains fermentate, the compound is then isolated and purified out from fermentate.Comprise the following steps that：
1 fermenting and producing
Cultivate microorganism conventional method, take penicillium oxalicum (Penicillium oxalicum) IBPT-6 be inoculated into PDA consolidate
Cultivate 2 to 3 days, be then seeded into nutrient solution in 28 DEG C of incubators on body slant medium, 28 DEG C of static gas wave refrigerators are after 30 days,
Obtain mycelium and zymotic fluid；The nutrient solution composition：Every liter of water contains the g of mannitol 20.0, the g of yeast extract 3.0, maltose 20.0
G, the g of monosodium glutamate 10.0, glucose 10.0 g, KH2PO4 0.5 g、MgSO4·7H2O 0.3 g、NaCl 15.0 g；
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.Mycelium acetone soln（Containing 20% ~ 30% water）Continuous ultrasound broken wall 3 times,
Filtering removes residue, obtains the mycelial crude extract containing acetone and water.Be concentrated under reduced pressure removal acetone, obtains the water-soluble of runic thing
Liquid, then with volume ratio 1:2, which add ethyl acetate, extracts 3 times, obtains ethyl acetate crude extract, is concentrated under reduced pressure near dry that mycelium soaks
The g of cream 36.5.
The separation and purification of 3 compounds
Mycelium medicinal extract is by 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol is gradient eluent, is subtracted
Press silica gel chromatograph column chromatography.By simple thin-layer chromatographic analysis, merge, be separated into component A-E.Component D (5.9 g) (two
Chloromethanes：Methanol v/v=100:1 eluate) with dichloromethane：Methanol is gradient elution agent, carries out pressured column silica gel
Analysis, merges after thin-layer chromatographic analysis and obtains five subfraction D1-D5.Component D2 (1.2 g) is with chloroform：Methanol=
1:2 be gradient elution agent, carries out gel filtration chromatography（Sephadex LH-20）, merge after thin-layer chromatographic analysis and obtain four
Subfraction D2-1 ~ D2-4.D2 subfraction D2-3 (212 mg) 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 55% acetonitrile contains 0.1% TFA, obtains shown compound (2.7
Mg, tR 13.8 min)。
The penicillium oxalicum (Penicillium oxalicum) IBPT-6, on December 25th, 2013 is deposited in
State's Type Tissue Collection, address:Wuhan Wuhan University, deposit number is：CCTCC NO：M 2013714.
The present invention also protects described compound answering in suppression human mouth epidermoid carcinoma cell proliferation is prepared
With, and application of the compound in anti-human oral cavity epidermoid carcinoma medicine is prepared.
The remarkable advantage of the present invention：The secalonic acid compound shown in research has no report and suppresses population with significant
Chamber epidermoid carcinoma cell-proliferation activity, has not yet to see report of the compound to human mouth epidermoid carcinoma cell inhibitory effect activity
Road, therefore in the market is also there is not yet medicine related to this.
Brief description of the drawings
COSY, HMBC and NOE signal main Fig. 1 Secalonic acid I.
Embodiment
The chemical constitution of signified compound in examples below：
The fermenting and producing and separation and purification of the compound of embodiment 1
1 fermenting and producing
Produce the fermented and cultured of bacterium：By culture microorganism conventional method, take penicillium oxalicum (Penicillium oxalicum)
IBPT-6 (is deposited in China typical culture collection center, address on December 25th, 2013:Wuhan Wuhan University, protects
Hiding numbering is：CCTCC NO：M 2013714) in right amount, it is inoculated into PDA solid slope culture mediums and is cultivated in 28 DEG C of incubators
2 to 3 days.
Take the inclined-plane culture penicillium oxalicum of 2 to 3 days (Penicillium oxalicum) appropriate IBPT-6, it is inoculated into dress
By 400mL nutrient solutions, [nutrient solution is constituted (g/l)：Mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, Portugal
Grape sugar 10.0, KH2PO40.5, MgSO4·7H2The constant volume of O 0.3, NaCl 15.0] 1000mL conical flasks in, 28 DEG C are static
After culture 30 days, mycelium and zymotic fluid are obtained.
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.Mycelium acetone soln（Containing 20% ~ 30% water）Continuous ultrasound broken wall 3 times,
Filtering removes residue, obtains the mycelial crude extract containing acetone and water.Be concentrated under reduced pressure removal acetone, obtains the water-soluble of runic thing
Liquid, then with volume ratio 1:2, which add ethyl acetate, extracts 3 times, obtains ethyl acetate crude extract, is concentrated under reduced pressure near dry that mycelium soaks
The g of cream 36.5.
The separation and purification of 3 compounds
Mycelium medicinal extract is by 100-200 mesh silica gel mixed samples, with petroleum ether：Dichloromethane：Methanol is gradient eluent, is subtracted
Press silica gel chromatograph column chromatography.By simple thin-layer chromatographic analysis, merge, be separated into component A-E.Component D (5.9 g) (two
Chloromethanes：Methanol v/v=100:1 eluate) with dichloromethane：Methanol is gradient elution agent, carries out pressured column silica gel
Analysis, merges after thin-layer chromatographic analysis and obtains five subfraction D1-D5.Component D2 (1.2 g) is with chloroform：Methanol=
1:2 be gradient elution agent, carries out gel filtration chromatography（Sephadex LH-20）, merge after thin-layer chromatographic analysis and obtain four
Subfraction D2-1 ~ D2-4.D2 subfraction D2-3 (212 mg) 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 55% acetonitrile contains 0.1% TFA, obtains shown compound (2.7
Mg, tR 13.8 min)。
It is yellow oil under compound normal temperature, high-resolution electrospray ionization mass spectrum HRESI-MS existsm/z：Given at 659.1377
Go out molecular ion peak [M+Na]+（calcd for C32H28NaO14, 659.1377）, it is 636 to point out molecular weight, with reference to wave spectrum letter
Breath speculates that molecular formula is C32H28O14。1H and13C-NMR data are shown in Table 1, and main COSY, HMBC and NOE signal is shown in Fig. 1.
The compound of table 11H and13C-NMR data (500 MHz1H and 126 MHz 13C, in DMSO-d 6 )
The test of the anti tumor activity in vitro of embodiment 2
1 laboratory sample and experimental method
The preparation test sample of sample solution is the pure compounds of separation and purification in above-mentioned implementation 1.Precision is weighed in right amount
Sample, the solution of concentration needed for being 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
Tetrazolium（MTT）Method is taken the logarithm the tumour cell in growth period, and cell density is adjusted into every milliliter 1 × 105Individual cell,
It is inoculated in by every 200 microlitres of hole in 96 porocyte culture plates, 5% CO is passed through in 37 DEG C2Incubator in cultivate 4 hours.Per hole
The sample liquid or blank solution of 2 microlitres of addition, after cultivating 24 hours, MTT liquid is added per hole（MTT every milliliter of 5 milligrams of physiology
Saline solution）10 microlitres, continue to cultivate 4 hours, 37 DEG C, 2000 revs/min centrifuge 8 minutes, draw supernatant.DMSO is added per hole
Each 100 microlitres, vibrate 15 minutes, after being completely dissolved to crystallization, produced using MD companies on micro oscillator
SPECTRAMAX Plus types ELIASA is determined per extinction of the hole at 570 nm（OD）Value.The sample in the orifice plate of same 96
Each concentration is respectively provided with three holes, the another blank control for setting three holes and acellular withered hole（If medicine has color to do accordingly
It is withered that drug concentration is acellular）.Each hole OD values first do corresponding acellular withered, then take three hole mean OD values by IR (%)=
(ODBlank control-ODSample)/ODBlank control× 100% calculates the proliferation inhibition rate of cell under each concentration（IR%）.
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 concentrations, using SPSS16.0 softwares
Carry out data processing and calculation of half inhibitory concentration IC50Value.It the results are shown in Table 2.
Inhibitory activity of the compound of table 2 to human mouth epiderm-like cancer cell multiplication
3. conclusion
The compound has preferable antitumor activity to human mouth epidermoid carcinoma cell.Can be thin as oral cavity epidermoid carcinoma is prepared
Born of the same parents' Proliferation Ability medicine or antineoplastic are used for the research of oral cavity epidermoid carcinoma.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (4)
1. compound。
2. the preparation method of compound as claimed in claim 1, it is characterised in that：Fermented and cultured penicillium oxalicum
(Penicillium oxalicum) IBPT-6, fermentate is obtained, the compound, institute are then isolated and purified out from fermentate
State penicillium oxalicum (Penicillium oxalicum) IBPT-6, it is deposited in Chinese Typical Representative culture on December 25th, 2013
Thing collection, address:Wuhan Wuhan University, deposit number is：CCTCC NO：M 2013714.
3. the compound described in claim 1 is preparing the application in suppressing human mouth epidermoid carcinoma cell proliferation.
4. application of the compound in anti-human oral cavity epidermoid carcinoma medicine is prepared described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710460060.5A CN107298669B (en) | 2017-06-17 | 2017-06-17 | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human oral epidermoid carcinoma |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710460060.5A CN107298669B (en) | 2017-06-17 | 2017-06-17 | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human oral epidermoid carcinoma |
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| Publication Number | Publication Date |
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| CN107298669A true CN107298669A (en) | 2017-10-27 |
| CN107298669B CN107298669B (en) | 2020-05-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201710460060.5A Active CN107298669B (en) | 2017-06-17 | 2017-06-17 | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human oral epidermoid carcinoma |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107298672A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | The secalonic acid I for coming from penicillium oxalicum is preparing the application of anti-human colon cancer drug |
| CN107353274A (en) * | 2017-06-17 | 2017-11-17 | 福州大学 | Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug |
| CN110407794A (en) * | 2019-04-26 | 2019-11-05 | 福州大学 | Secalonic acid K derived from penicillium oxalicum and the application on inhibition cancer cell multiplication |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102408997A (en) * | 2010-11-29 | 2012-04-11 | 国家海洋局第三海洋研究所 | Deep-sea-sourced penicillium F11 capable of producing compound secalonic acid F with cytotoxic activity |
| CN107353274A (en) * | 2017-06-17 | 2017-11-17 | 福州大学 | Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug |
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2017
- 2017-06-17 CN CN201710460060.5A patent/CN107298669B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102408997A (en) * | 2010-11-29 | 2012-04-11 | 国家海洋局第三海洋研究所 | Deep-sea-sourced penicillium F11 capable of producing compound secalonic acid F with cytotoxic activity |
| CN107353274A (en) * | 2017-06-17 | 2017-11-17 | 福州大学 | Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug |
Non-Patent Citations (2)
| Title |
|---|
| GURU,SANTOSH KUMAR;等人: "Secalonic Acid-D Represses HIF1α/VEGF-Mediated Angiogenesis by Regulating the Akt/mTOR/p70S6K Signaling Cascade", 《CANCER RESEARCH》 * |
| ISHIDA,TORAO: "Preparation and anti-microbial, anti-tumor properties of substance 25025 (secalonic acid D)", 《SUZUKA IRYO KAGAKU GIJUTSU DAIGAKU KIYO》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107298672A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | The secalonic acid I for coming from penicillium oxalicum is preparing the application of anti-human colon cancer drug |
| CN107353274A (en) * | 2017-06-17 | 2017-11-17 | 福州大学 | Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug |
| CN107353274B (en) * | 2017-06-17 | 2020-05-08 | 福州大学 | Selenolonic acid I from penicillium oxalicum and application thereof in preparation of human esophageal cancer resistant medicine |
| CN110407794A (en) * | 2019-04-26 | 2019-11-05 | 福州大学 | Secalonic acid K derived from penicillium oxalicum and the application on inhibition cancer cell multiplication |
| CN110407794B (en) * | 2019-04-26 | 2022-10-14 | 福州大学 | Selenolonic acid K derived from penicillium oxalicum and application thereof in inhibiting cancer cell proliferation |
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