CN107485607A - The secalonic acid H for coming from penicillium oxalicum is preparing the application of anti-human oesophagus cancer drug - Google Patents
The secalonic acid H for coming from penicillium oxalicum is preparing the application of anti-human oesophagus cancer drug Download PDFInfo
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Abstract
The present invention relates to the secalonic acid H for coming from penicillium oxalicum to prepare the application of anti-human oesophagus cancer drug.The compound, which has, suppresses esophagus carcinoma proliferation effect, and 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 esophagus cancer cell EC9706.Can be as the research for preparing human esophagus cancer cell Proliferation Ability medicine or antineoplastic and being used for human esophagus cancer.
Description
Technical field
The present invention relates to the secalonic acid H for coming from penicillium oxalicum to prepare the application of anti-human oesophagus cancer drug, belongs to medicine
Field.
Background technology
Secalonic acid class compound（Secalonic acids）Belong to ergot pigment（Ergochrome）Secondary metabolism produces
Thing, it 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
Cancer, in the range of 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 are extra large
Foreign fungi can produce the secalonic acid class compound that structure is novel, activity is good during cometabolism, have 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 are：CCTCC NO：
M 2013714) the crude extract of tunning have good cell inhibitory effect activity, its active component is ground then
Study carefully.Research find shown in secalonic acid class compound there is anti-human cancer of the esophagus activity, have not yet to see the compound to people's oesophagus
The report of the proliferation inhibition activity of cancer 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 the secalonic acid H for coming from penicillium oxalicum to prepare answering for anti-human oesophagus cancer drug
With.The compound, which has, suppresses human esophagus cancer cell proliferation function, has anti-human cancer of the esophagus activity.Its structural formula is：
。
The preparation method of the compound, be by fermented and cultured penicillium oxalicum (Penicillium oxalicum)
IBPT-6, fermentate is obtained, 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 is inoculated into PDA and consolidates
To 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 is containing 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,
Filter off and remove residue, obtain the mycelial crude extract containing acetone and water.It is concentrated under reduced pressure and removes 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, merge after thin-layer chromatographic analysis and obtain 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 (2.4
Mg, tR 8.1 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 are：CCTCC NO：M 2013714.
The present invention also protects described compound preparing the application in suppressing human esophagus cancer cell hyperproliferation agent, and should
Application of the compound in anti-human oesophagus cancer drug is prepared.
The remarkable advantage of the present invention：The secalonic acid compound shown in research has no report and suppresses people's food with significant
Pipe cancer proliferation activity, have not yet to see report of the compound to human esophagus cancer cell proliferation inhibition activity, therefore in the market
There is not yet medicine related to this.
Brief description of the drawings
COSY, HMBC and NOE signal main Fig. 1 Secalonic acid H.
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, protect
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
3 days.
Take the inclined-plane culture penicillium oxalicum of 3 days (Penicillium oxalicum) appropriate IBPT-6, it is inoculated into and is equipped with
[nutrient solution forms (g/l) to 400mL nutrient solutions：Mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, grape
Sugar 10.0, KH2PO40.5, MgSO4·7H2The constant volume of O 0.3, NaCl 15.0] 1000mL conical flasks in, 28 DEG C of static trainings
After supporting 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 30% water）Continuous ultrasound broken wall 3 times, filtering
Residue is removed, obtains the mycelial crude extract containing acetone and water.It is concentrated under reduced pressure and removes acetone, obtains the aqueous solution of runic thing,
Again with volume ratio 1:2, which add ethyl acetate, extracts 3 times, obtains ethyl acetate crude extract, is concentrated under reduced pressure into and closely does to obtain mycelium medicinal extract
36.5 g。
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, merge after thin-layer chromatographic analysis and obtain 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 (2.4
Mg, tR 8.1 min)。
It is yellow oil under compound normal temperature, high-resolution electrospray ionization mass spectrum HRESI-MS existsm/z：Given at 659.1370
Go out molecular ion peak [M+Na]+（calcd for C32H28NaO14, 659.1377）, it is 636 to prompt molecular weight, is believed with reference to wave spectrum
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 weighs in right amount
Sample, the solution of concentration needed for methanol is configured to, 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
2 microlitres of sample liquid or blank solution is added, after cultivating 24 hours, MTT liquid is added per hole（MTT every milliliter of 5 milligrams of physiology
Saline solution）10 microlitres, continue culture 4 hours, 37 DEG C, 2000 revs/min centrifuge 8 minutes, draw supernatant.DMSO is added per hole
Each 100 microlitres, vibrated 15 minutes on micro oscillator, after being completely dissolved to crystallization, utilize the production of MD companies
SPECTRAMAX Plus types ELIASA measure is 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 it is 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.
The inhibitory activity that the compound of table 2 is bred to human esophagus cancer cell
3. conclusion
The compound has preferable antitumor activity to human esophagus cancer cell.It can be used as and prepare human esophagus cancer cell Proliferation Ability
Medicine or antineoplastic are used for the research of human esophagus cancer.
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 are：CCTCC NO：M 2013714.
3. compound described in claim 1 is preparing the application in suppressing human esophagus cancer cell hyperproliferation agent.
4. application of the compound in anti-human oesophagus cancer drug is prepared described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710460064.3A CN107485607B (en) | 2017-06-17 | 2017-06-17 | Application of seclenic acid H derived from penicillium oxalicum in preparation of human esophageal cancer resistant medicine |
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| CN201710460064.3A CN107485607B (en) | 2017-06-17 | 2017-06-17 | Application of seclenic acid H derived from penicillium oxalicum in preparation of human esophageal cancer resistant medicine |
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| CN107485607A true CN107485607A (en) | 2017-12-19 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107298671A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from the secalonic acid H of penicillium oxalicum and prepare the application of anti-human colon cancer drug |
| CN107298670A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from penicillium oxalicum secalonic acid H and prepare anti-human oral cavity epidermoid carcinoma medicinal application |
| CN110922383A (en) * | 2018-12-04 | 2020-03-27 | 福州大学 | iso-Penicillium xanthone A from Penicillium oxalicum and its application in gastric cancer |
| CN110922378A (en) * | 2018-12-04 | 2020-03-27 | 福州大学 | iso-Penicillium xanthone A from penicillium oxalicum and application in nasopharyngeal carcinoma |
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| 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 |
| CN107298670A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from penicillium oxalicum secalonic acid H and prepare anti-human oral cavity epidermoid carcinoma medicinal application |
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2017
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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 |
| CN107298670A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from penicillium oxalicum secalonic acid H and prepare anti-human oral cavity epidermoid carcinoma medicinal application |
Non-Patent Citations (2)
| Title |
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| 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》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107298671A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from the secalonic acid H of penicillium oxalicum and prepare the application of anti-human colon cancer drug |
| CN107298670A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from penicillium oxalicum secalonic acid H and prepare anti-human oral cavity epidermoid carcinoma medicinal application |
| CN107298670B (en) * | 2017-06-17 | 2020-05-08 | 福州大学 | Application of medicine derived from penicillium oxalicum seclenum ketonic acid H in preparation of anti-human oral epidermoid carcinoma medicines |
| CN110922383A (en) * | 2018-12-04 | 2020-03-27 | 福州大学 | iso-Penicillium xanthone A from Penicillium oxalicum and its application in gastric cancer |
| CN110922378A (en) * | 2018-12-04 | 2020-03-27 | 福州大学 | iso-Penicillium xanthone A from penicillium oxalicum and application in nasopharyngeal carcinoma |
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