CN109134416A - Secalonic acid H derived from penicillium oxalicum is in the application for preparing human cervical cancer 1 cancer drug - Google Patents
Secalonic acid H derived from penicillium oxalicum is in the application for preparing human cervical cancer 1 cancer drug Download PDFInfo
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- CN109134416A CN109134416A CN201710460063.9A CN201710460063A CN109134416A CN 109134416 A CN109134416 A CN 109134416A CN 201710460063 A CN201710460063 A CN 201710460063A CN 109134416 A CN109134416 A CN 109134416A
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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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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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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 H derived from penicillium oxalicum in the application for preparing human cervical cancer 1 cancer drug.The compound has cervical cancer inhibiting cel l proliferation.Its structural formula are as follows:.By fermented and cultured penicillium oxalicum (Penicillium oxalicum) IBPT-6, 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 cervical carcinoma cell Hela.It can be used as the research for preparing human cervical carcinoma cell Proliferation Ability drug or anti-tumor drug for human cervical carcinoma.
Description
Technical field
The present invention relates to the secalonic acid H derived from penicillium oxalicum in the application for preparing human cervical cancer 1 cancer drug, belongs to medicine
Field.
Background technique
Secalonic acid class compound (Secalonic acids) belongs to the production of ergot pigment (Ergochrome) secondary metabolism
Object is xanthone dimer.Since Stoll etc. was in the isolated secalonic acid A from fungi in 1952
After (Secalonic acid A), the compound secalonic acid (A, B, C, D, E, F, G) of the series just constantly by
It was found that and research.Secalonic acid class compound has a variety of different physiological activity, with secalonic acid D(Secalonic acid
D, SAD) for, the SAD of 5 mg/ml is added in physiological saline, within the scope of 5-20 mg, it can treatment early stage bladder
Cancer, within the scope of 50-100 mg, and be free from side effects generation effective in cure to more serious bladder cancer.It has been investigated that Yi Xiehai
Foreign fungi can generate structure novel, activity good secalonic acid class compound during cometabolism, have good medicine
With and industrialization prospect.
The present inventor studies and learns, penicillium oxalicum (Penicillium oxalicum) IBPT-6, (in 2013 12
The moon is deposited in China typical culture collection center on 25th, and address: Wuhan Wuhan University, deposit number are: CCTCC NO:M
2013714) crude extract of tunning has good cell inhibitory effect activity, studies then its active constituent.
Research finds that shown secalonic acid class compound has anti-human cervix cancer activity, has not yet to see the compound to human cervical cancer 1
The report of the proliferation inhibition activity of cancer cell, therefore in the market also there is not yet drug related to this.
Summary of the invention
The purpose of the present invention is to provide the secalonic acid H derived from penicillium oxalicum to prepare answering for human cervical cancer 1 cancer drug
With.The compound, which has, inhibits human cervical carcinoma cell proliferation function, has anti-human cervix cancer activity.Its structural formula are as follows:
。
The preparation method of the compound, be by fermented and cultured penicillium oxalicum (Penicillium oxalicum)
IBPT-6 obtains fermentation material, the compound is then isolated and purified out from fermentation material.Specific step is as follows:
1 fermenting and producing
Cultivate microorganism conventional method, take penicillium oxalicum (Penicillium oxalicum) to be inoculated into PDA solid by IBPT-6
It cultivates 2 to 3 days, is then seeded into culture solution in 28 DEG C of incubators on body slant medium, 28 DEG C after static gas wave refrigerator 30 days,
Obtain mycelium and fermentation liquid；The culture solution composition: every liter of water contains 20.0 g of mannitol, 3.0 g of yeast extract, maltose 20.0
G, 10.0 g of monosodium glutamate, 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 with acetone soln (contain 20% ~ 30% water) continuous ultrasound broken wall 3 times,
Filtering removal residue, obtains the mycelial crude extract containing acetone and water.Removal acetone is concentrated under reduced pressure, obtains the water-soluble of runic object
Liquid, then ethyl acetate is added with volume ratio 1:2 and is extracted 3 times, ethyl acetate crude extract is obtained, is concentrated under reduced pressure into close dry that mycelium soaks
36.5 g of cream.
The separation and purification of 3 compounds
Mycelium medicinal extract is by 100-200 mesh silica gel mixed sample, and using petroleum ether: methylene chloride: methanol is subtracted as gradient eluent
Press silica gel chromatographic column chromatography.By simple thin-layer chromatographic analysis, merges, be separated into component A-E.Component D (5.9 g) (two
Chloromethanes: the eluate of methanol v/v=100:1) using methylene chloride: methanol carries out pressured column silica gel as gradient elution agent
Analysis merges after thin-layer chromatographic analysis and obtains five subfraction D1-D5.Component D2 (1.2 g) is with chloroform: methanol=
1:2 is gradient elution agent, is carried out gel filtration chromatography (Sephadex LH-20), merges after thin-layer chromatographic analysis and obtains four
Subfraction D2-1 ~ D2-4.The subfraction D2-3 (212 mg) of D2 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, it has been deposited on December 25th, 2013
State's Type Tissue Collection, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013714.
The present invention also protects the compound to inhibit the application in human cervical carcinoma cell hyperproliferation agent in preparation, and
The compound is preparing the application in anti-human cervix cancer drug.
Remarkable advantage of the invention: the secalonic acid compound shown in studying has not been reported and has significant inhibition people
Cervical carcinoma proliferation activity has not yet to see the compound to the report of human cervical carcinoma cell proliferation inhibition activity, therefore market
On also there is not yet drug related to this.
Detailed description of the invention
Fig. 1 Secalonic acid main COSY of H, 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 penicillium oxalicum (Penicillium oxalicum)
IBPT-6 (be deposited in China typical culture collection center on December 25th, 2013, address: protect by Wuhan Wuhan University
Hiding number is: CCTCC NO:M 2013714) in right amount, being inoculated into PDA solid slope culture medium and cultivates in 28 DEG C of incubators
3 days.
Take 3 days penicillium oxalicums of inclined-plane culture (Penicillium oxalicum) appropriate IBPT-6, it is inoculated into and is equipped with
[culture solution forms (grams per liter) to 400mL culture solution: mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, glucose
10.0 KH2PO40.5, MgSO4·7H215.0 constant volume of O 0.3, NaCl] 1000mL conical flask in, 28 DEG C of static gas wave refrigerators
After 30 days, mycelium and fermentation liquid are obtained.
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.Mycelium is filtered with acetone soln (containing 30% water) continuous ultrasound broken wall 3 times
Residue is removed, the mycelial crude extract containing acetone and water is obtained.Removal acetone is concentrated under reduced pressure, obtains the aqueous solution of runic object,
Ethyl acetate is added with volume ratio 1:2 again to extract 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 sample, and using petroleum ether: methylene chloride: methanol is subtracted as gradient eluent
Press silica gel chromatographic column chromatography.By simple thin-layer chromatographic analysis, merges, be separated into component A-E.Component D (5.9 g) (two
Chloromethanes: the eluate of methanol v/v=100:1) using methylene chloride: methanol carries out pressured column silica gel as gradient elution agent
Analysis merges after thin-layer chromatographic analysis and obtains five subfraction D1-D5.Component D2 (1.2 g) is with chloroform: methanol=
1:2 is gradient elution agent, is carried out gel filtration chromatography (Sephadex LH-20), merges after thin-layer chromatographic analysis and obtains four
Subfraction D2-1 ~ D2-4.The subfraction D2-3 (212 mg) of D2 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 room temperature, high-resolution electrospray ionization mass spectrum HRESI-MS existsm/z: it is given at 659.1370
Molecular ion peak [M+Na] out+(calcd for C32H28NaO14, 659.1377), prompting molecular weight is 636, is believed in conjunction with 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.
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-mentioned implementation 1.Precision weighs in right amount
Sample is configured to the solution of required concentration with 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 density5A cell,
It is inoculated in 96 porocyte culture plates for 200 microlitres by every hole, is passed through 5% CO in 37 DEG C2Incubator in cultivate 4 hours.Every hole
2 microlitres of sample liquid or blank solution is added, after culture 24 hours, MTT liquid (every milliliter of 5 milligrams of physiology of MTT are added in every hole
Saline solution) 10 microlitres, continue culture 4 hours, 37 DEG C, 2000 revs/min are centrifuged 8 minutes, draw supernatant.DMSO is added in every hole
It each 100 microlitres, is vibrated 15 minutes on micro oscillator, until utilizing the production of MD company after crystallization is completely dissolved
SPECTRAMAX Plus type microplate reader measures extinction (OD) value of every hole at 570 nm.The sample in 96 orifice plate of same
Each concentration is respectively provided with three holes, and the another blank control that three holes are arranged and cell-free withered hole are (if drug has color to do accordingly
It is withered that drug concentration is cell-free).Each hole OD value first do it is corresponding cell-free withered, then take 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
Carry out data processing and calculation of half inhibitory concentration IC50Value.It the results are shown in Table 2.
The inhibitory activity that 2 compound of table is proliferated human cervical carcinoma cell
3. conclusion
The compound has preferable anti-tumor activity to human cervical carcinoma cell.It can be used as and prepare human cervical carcinoma cell proliferation
Drug or anti-tumor drug is inhibited to be used for the research of human cervical carcinoma.
Claims (4)
1. compound。
2. the preparation method of compound as described in claim 1, it is characterised in that: fermented and cultured penicillium oxalicum
(Penicillium oxalicum) IBPT-6, fermentation material is obtained, the compound, institute are then isolated and purified out from fermentation material
State penicillium oxalicum (Penicillium oxalicum) IBPT-6, Chinese Typical Representative culture is deposited on December 25th, 2013
Object collection, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013714.
3. compound described in claim 1 inhibits the application in human cervical carcinoma cell hyperproliferation agent in preparation.
4. compound described in claim 1 is preparing the application in anti-human cervix cancer drug.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710460063.9A CN109134416B (en) | 2017-06-17 | 2017-06-17 | Application of seclenic acid H derived from penicillium oxalicum in preparation of human cervical cancer drugs |
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| CN201710460063.9A CN109134416B (en) | 2017-06-17 | 2017-06-17 | Application of seclenic acid H derived from penicillium oxalicum in preparation of human cervical cancer drugs |
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| CN109134416A true CN109134416A (en) | 2019-01-04 |
| CN109134416B CN109134416B (en) | 2022-03-15 |
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Cited By (2)
| 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4418061A (en) * | 1981-04-16 | 1983-11-29 | Asahi Kasei Kogyo Kabushiki Kaisha | Secalonic acid derivatives and method for preparing thereof |
| 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 |
| CN107298671B (en) * | 2017-06-17 | 2020-05-08 | 福州大学 | Selenolonic acid H from penicillium oxalicum and application thereof in preparing medicine for resisting human colon cancer |
-
2017
- 2017-06-17 CN CN201710460063.9A patent/CN109134416B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4418061A (en) * | 1981-04-16 | 1983-11-29 | Asahi Kasei Kogyo Kabushiki Kaisha | Secalonic acid derivatives and method for preparing thereof |
| 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 |
| CN107298671B (en) * | 2017-06-17 | 2020-05-08 | 福州大学 | Selenolonic acid H from penicillium oxalicum and application thereof in preparing medicine for resisting human colon cancer |
Non-Patent Citations (3)
| Title |
|---|
| RAYMOND ANDERSEN等: "Secalonic Acids D and F Are Toxic Metabolites of Aspergillus", 《J. ORG. CHEM.》 * |
| 徐帮等: "中华剑角蝗共生真菌Penicillium oxalicum中苯并吡喃酮二聚体类化合物的研究", 《天然产物研究与开发》 * |
| 陈立等: "海绵来源真菌代谢产物中抗肿瘤活性成分研究", 《中国海洋大学学报》 * |
Cited By (3)
| 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 |
| 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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