CN102260271B - Cytochalasin compound as well as preparation method thereof and purpose - Google Patents
Cytochalasin compound as well as preparation method thereof and purpose Download PDFInfo
- Publication number
- CN102260271B CN102260271B CN 201110153240 CN201110153240A CN102260271B CN 102260271 B CN102260271 B CN 102260271B CN 201110153240 CN201110153240 CN 201110153240 CN 201110153240 A CN201110153240 A CN 201110153240A CN 102260271 B CN102260271 B CN 102260271B
- Authority
- CN
- China
- Prior art keywords
- compound
- preparation
- cell
- zhn1
- chloroform
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a preparation method and a purpose of a cytochalasin compound. According to the invention, a novel cytochalasin compound is produced by using Aspergillus flavipes ZHN1-09 in a Guangxi mangrove forest plant Cerbera manghas root mug sample. It is proved by experiments that the compound can be used as cell proliferation inhibitors or antitumor agents.
Description
Technical field:
The present invention relates to that ((on March 24th, 2011 was deposited in Wuhan University Chinese Typical Representative culture collection center, and deposit number is: the method for CCTCC M 2011069) producing the cytochalasin compounds with yellow bright aspergillus ZHN1-09 (Aspergillus flavipes ZHN1-09); The invention still further relates to the purposes of this compounds in preparation inhibition of cell proliferation or antineoplastic agent.
Background technology:
Cytochalasin is the relevant secondary metabolite of a kind of structure and activity of being produced by fungi, and scientists finds that this compounds has many unusual and interesting effects to cell.Inventor's research is learnt, (deposit number is: CCTCC M 2011069) crude extract of liquid fermentation production after ultrasonication has good cell inhibitory effect active to yellow bright aspergillus ZHN1-09 (Aspergillus flavipes ZHN1-09), then its activeconstituents is studied.Compound had anti-tumor activity shown in research was found, had not yet to see the report of chemical structure and the cell inhibitory effect activity of this compound, so also there is not yet relevant therewith medicine on the market.
Summary of the invention:
The present invention aims to provide one and has inhibition tumor cell propagation, has the new compound of anti-tumor activity.Its structural formula is
Its constitutional features is: this compound is the cytochalasin of new ring-type.
Compound of the present invention can cultivate to obtain the fermented product that is rich in the cytochalasin compounds by microbial fermentation, then adopts the method separation and purification such as silica gel column chromatography, Sephadex LH-20 gel filtration chromatography, RP-18 reversed-phase silica gel column chromatography and half preparative HPLC to obtain from fermented product.
Enumerated among the following embodiment of the present invention utilize yellow bright aspergillus ZHN1-09 (Aspergillus flavipes ZHN1-09) (deposit number is: CCTCC M 2011069) preparation the compounds of this invention example.
Embodiment:
The chemical structure of the compound of indication in following embodiment (Arabic numerals in the structural formula are marks of carbon atom in the chemical structure) is:
Fermentative production and the separation and purification of embodiment 1 this compound
1 fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, (deposit number is: CCTCC M 2011069) an amount of to get yellow bright aspergillus ZHN1-09 (Aspergillus flavipesZHN1-09), be inoculated on the PDA solid slant culture base, in 28 degrees centigrade of incubators, cultivated 6 days.
It is an amount of to get 6 days yellow bright aspergillus ZHN1-09 (Aspergillus flavipes) of slant culture, is inoculated into 300mL nutrient solution [substratum composition (grams per liter): glucose 10.0, maltose 20.0, N.F,USP MANNITOL 20.0, monosodium glutamate 10.0, KH is housed
2PO
40.5, MgSO
40.3, yeast extract paste 3.0, Semen Maydis powder 0.3, pH=6.5] the 1000mL Erlenmeyer flask in, leave standstill at 28 ℃ and to cultivate 40 days, obtain mycelium and fermented liquid.
The acquisition of 2 medicinal extract
With gauze with mycelium and separation of fermentative broth.With fermented liquid equal-volume ethyl acetate extraction three times, extraction liquid is evaporated to dried; Bacterial cell disruption with 80% acetone extract 3 times, is evaporated to and does not contain acetone, and the residue water layer is with equal-volume ethyl acetate extraction 3 times, and extraction liquid is evaporated to dried; Merge the extract of thalline and fermented liquid, get crude extract, totally 3.4 grams.
The separation and purification of 3 compounds
After medicinal extract dissolves with the chloroform-methanol mixed solvent, add 100-200 order silica gel (Qingdao Haiyang Chemical Industry Group Corp.'s product) and mix sample, after the removal of solvent under reduced pressure, use silica gel column chromatography, with sherwood oil, sherwood oil-acetone, chloroform, chloroform-methanol are that solvent carries out gradient elution, are divided into 4 cuts (Fr-1, Fr-2, Fr-3 and Fr-4).Fr-2 (20: 1 eluates of chloroform-methanol), successively carry out the LH-20 column chromatography take chloroform-methanol (1: 1) as solvent, again through the RP-18 reversed-phase silica gel column chromatography take methyl alcohol: water (40: 60) carries out wash-out as solvent, finally by partly preparing RPLC preparation (methyl alcohol: water=70: 30) must this compound.
Compound white amorphous solid, [α]
25 D=-5.5 (c 0.1, MeOH), and molecular formula C
28H
33NO
6, HR-ESI-MS m/z:480.2379[M+H]
+, calculated value 480.2386.IR?v
maxcm
-1(KBr)：3420，2925，2866，1741，1716，1702，1459，1078，665，573。
1H and
13The C-NMR data see Table 1.
Table 1 compound
1H (600MHz) and
13The NMR data of C (150MHz) in CDCl3
a
A) this table signal ownership is based on DEPT, HMQC and HMBC spectrum analysis result.The multiple degree binding molecule amount of carbon signal is determined.
B) numeral and the code name in this hurdle represents respectively
1H-
1In the H COSY spectrum with corresponding line in
1H provides the coincidence signal
1H nuclear.
C) numeral in this hurdle and code name represent respectively in HMBC spectrum with corresponding line in
1H provides the coincidence signal
13C nuclear.
The test of embodiment 2 anti tumor activity in vitro
The cell inhibitory effect active testing
1 laboratory sample and experimental technique
The preparation specimen of sample solution is the pure compounds of separation and purification in above-described embodiment 1.Precision takes by weighing an amount of sample, is mixed with the solution of desired concn with methyl alcohol, and is active for surveying.
The succeeding transfer culture of clone and cell adopts the cancerous cell lines such as people's lung cancer A549 cell, human leukemia HL60 cell and P 388 cells.Various cells are all with the RPMI-1640 substratum that contains 10%FBS, at 37 ℃ of succeeding transfer culture in the incubator that passes into 5% carbonic acid gas.
The cell inhibitory effect activity test method
It is every milliliter 2 * 10 that people's lung cancer A549 cell that Sulforhodamine B (SRB) method is taken the logarithm vegetative period is mixed with density with fresh RPMI-1640 substratum
5The cell suspension of individual cell is inoculated in 96 orifice plates by every hole 200 microlitres, and every hole adds sample or the blank solution of 2 microlitre different concns, 37 ℃ of lower cultivations 24 hours.Get it filled cell after cultivating under the thing effect, the morphological change that at first causes in optical microphotograph Microscopic observation drug treating is judged to have or not the cell cycle to suppress the morphological feature of apoptosis or necrocytosis, then 4 ℃, 3000 rev/mins centrifugal 3 minutes, suck supernatant.Add 20% Tricholroacetic Acid, 50 microlitres in every porocyte, place 4 ℃ to fix 1 hour, water flushing 5 times and dry air.Every hole adds acetum 50 microlitres of 0.4%SRB and left standstill 30 minutes in room temperature.Clean 4 times with 1% acetic acid water, remove unconjugated free SRB dyestuff.Every hole adds 150 microlitre Tris damping fluids (10mmol/L, pH 10.5) soluble protein combination dye and utilizes MD company to produce SPECTRAMAX Plus type microplate reader and measure every hole in optical density(OD) (OD) value at 520nm place.Each concentration of sample all arranges three holes in same 96 orifice plates, and other establishes three hole blanks and acellular zeroing hole (if medicine has color will do the acellular zeroing of relative medicine concentration).Each hole OD value is done first corresponding acellular zeroing, gets the average OD value in three holes by IR%=(OD again
Blank-OD
Sample)/OD
BlankThe X100% formula is calculated the cell proliferation inhibition rate (IR%) under each concentration.
The human leukemia HL60 cell that tetrazolium (MTT) method is taken the logarithm vegetative period and murine leukemia P388 cell transfer to every milliliter 2 * 10 with cell density
5Individual cell is inoculated in the 96 porocyte culture plates by every hole 200 microlitres, passes into 5%CO in 37 ℃
2Incubator in cultivated 4 hours.Every hole adds each 2 microlitre of sample liquid or blank solution, cultivates after 24 hours, and every hole adds MTT liquid (every milliliter of 5 milligrams of normal saline solutions of MTT) 10 microlitres, continues cultivation 4 hours, 37 ℃, 2000 rev/mins centrifugal 8 minutes, suck supernatant.Every hole adds each 100 microlitre of DMSO, vibrates 15 minutes at micro oscillator, after dissolving fully to crystallization, utilizes MD company to produce SPECTRAMAX Plus type microplate reader and measures every hole at the light absorption value (OD value) at 570nm place.Each concentration of sample all arranges three holes in same 96 orifice plates, and other establishes three hole blanks and acellular zeroing hole (if medicine has color will do the acellular zeroing of relative medicine concentration).Each hole OD value is done first corresponding acellular zeroing, gets the average OD value in three holes by IR%=(OD again
Blank-OD
Sample)/OD
BlankThe X100% formula is calculated the cell proliferation inhibition rate (IR%) under each concentration.
2. experimental result
Cell inhibitory effect active testing result
In srb assay or mtt assay test, this compound of different concns suppresses to the results are shown in Table 2 to the propagation of people's lung cancer A549 cell, human leukemia HL60 cell and P 388 cells.
Table 2 compound is active to the inhibition of different carcinoma cell proliferation
3 conclusions
This compound has obvious kinds of tumor cells inhibited proliferation, can be used as the research that inhibition of cell proliferation or antineoplastic agent are used for cancer therapy drug.
Claims (5)
1. compound structure is shown below
2. the preparation method of the described compound of claim 1 is characterized in that the yellow bright aspergillus ZHN1-09 (Aspergillusflavipes, ZHN1-09) of fermentation culture, obtains the fermented product that contains above-claimed cpd, and then separation and purification goes out this compound from fermented product.
3. preparation method claimed in claim 2, wherein with described fermented product through silica gel column chromatography, carry out gradient elution take sherwood oil, sherwood oil-acetone, chloroform, chloroform-methanol as solvent, 20: 1 wash-out parts of chloroform-methanol, through Sephadex LH-20 gel filtration chromatography, RP-18 reversed-phase silica gel column chromatography and half preparative HPLC separation and purification must this compounds again.
4. compound claimed in claim 1 is in the purposes of preparation in the inhibition of cell proliferation.
5. compound claimed in claim 1 is in the purposes of preparation in the antitumor drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110153240 CN102260271B (en) | 2011-06-01 | 2011-06-01 | Cytochalasin compound as well as preparation method thereof and purpose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110153240 CN102260271B (en) | 2011-06-01 | 2011-06-01 | Cytochalasin compound as well as preparation method thereof and purpose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102260271A CN102260271A (en) | 2011-11-30 |
| CN102260271B true CN102260271B (en) | 2013-10-30 |
Family
ID=45007132
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110153240 Expired - Fee Related CN102260271B (en) | 2011-06-01 | 2011-06-01 | Cytochalasin compound as well as preparation method thereof and purpose |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102260271B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103787953B (en) * | 2014-01-17 | 2015-12-02 | 杭州维康科技有限公司 | Compd A spochalasin V and its preparation method and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003006060A1 (en) * | 2001-07-09 | 2003-01-23 | Kyowa Hakko Kogyo Co., Ltd. | Sh3 domain binding inhibitors |
| CN101416962A (en) * | 2008-12-05 | 2009-04-29 | 中国热带农业科学院热带生物技术研究所 | Use of cytochalasin D in preparing anti-tumor medicine |
| CN101591288A (en) * | 2009-06-26 | 2009-12-02 | 中国科学院海洋研究所 | Cytochalasin compounds and its production and use |
-
2011
- 2011-06-01 CN CN 201110153240 patent/CN102260271B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003006060A1 (en) * | 2001-07-09 | 2003-01-23 | Kyowa Hakko Kogyo Co., Ltd. | Sh3 domain binding inhibitors |
| CN101416962A (en) * | 2008-12-05 | 2009-04-29 | 中国热带农业科学院热带生物技术研究所 | Use of cytochalasin D in preparing anti-tumor medicine |
| CN101591288A (en) * | 2009-06-26 | 2009-12-02 | 中国科学院海洋研究所 | Cytochalasin compounds and its production and use |
Non-Patent Citations (2)
| Title |
|---|
| Takamatsu, S，等.Characterization of Mycotypha metabolites found to be inhibitors of cell adhesion molecules.《JOURNAL OF ANTIBIOTICS 》.2002,第55卷(第6期),第585-592页. * |
| Tomoda, H,等.Structure elucidation of fungal phenochalasins, novel inhibitors of lipid droplet formation in mouse macrophages.《JOURNAL OF ANTIBIOTICS》.1999,第52卷(第10期),第857-861页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102260271A (en) | 2011-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103865808B (en) | A kind of anticancer usage of the penicillium sp enol A1 coming from Aspergillus citrimum | |
| CN107298671A (en) | Come from the secalonic acid H of penicillium oxalicum and prepare the application of anti-human colon cancer drug | |
| CN107353274A (en) | Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug | |
| CN104402899B (en) | Citrinin compounds penicitrinol L coming from Aspergillus citrimum and its preparation method and application | |
| CN107298672A (en) | The secalonic acid I for coming from penicillium oxalicum is preparing the application of anti-human colon cancer drug | |
| CN101735236B (en) | Dimer citrinin compounds and preparation and use thereof | |
| CN107298670A (en) | Come from penicillium oxalicum secalonic acid H and prepare anti-human oral cavity epidermoid carcinoma medicinal application | |
| CN101812079B (en) | Piprazine compound containing polysulfide bond and preparation method and application thereof | |
| CN107485607A (en) | The secalonic acid H for coming from penicillium oxalicum is preparing the application of anti-human oesophagus cancer drug | |
| CN104592082B (en) | Come from the penicillium sp enol D of Aspergillus citrimum2preparation method and applications | |
| CN107298669A (en) | Come from the secalonic acid I of penicillium oxalicum and anti-human oral cavity epidermoid carcinoma medicinal application | |
| CN104387396B (en) | Come from indole terpene speradine E and the application of aspergillus oryzae | |
| CN102260271B (en) | Cytochalasin compound as well as preparation method thereof and purpose | |
| CN110407792A (en) | Derived from the secalonic acid class compound Secalonic acid J and preparation method of penicillium oxalicum | |
| CN102268005B (en) | Indole diketopiperazine alkaloid compound derived from tryptophan and proline and preparation method and application thereof | |
| CN103058846B (en) | Benzoquinone derivative from aspergillus aculeatus and application of benzoquinone derivative | |
| CN103030683B (en) | Peptaibol and preparation method and use thereof | |
| CN104370917A (en) | Indole terpene speradine H derived from aspergillus oryzae and application | |
| CN101735193B (en) | 9-anthranone spiro lactone compound and preparation method and application thereof | |
| CN103012559A (en) | Peptaibol compound and application thereof | |
| CN103058971B (en) | Benzoquinone spiro compound derived from aspergillus aculeatus and application of benzoquinone spiro compound | |
| CN102276613B (en) | Sesquiterpene alkaloid compound as well as preparation method and application thereof | |
| CN101735237B (en) | Trimeric citrinin compounds as well as preparation methods and applications thereof | |
| CN104211630B (en) | A kind of alkaloid compound and application thereof that comes from tangerine green trichoderma | |
| CN105037397A (en) | Sulfur-bridge-containing complex-ring-series alkaloids compound, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131030 Termination date: 20140601 |