CN104211780B - A kind of cyclic depsipeptides compound and its production and use - Google Patents
A kind of cyclic depsipeptides compound and its production and use Download PDFInfo
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- CN104211780B CN104211780B CN201310211876.6A CN201310211876A CN104211780B CN 104211780 B CN104211780 B CN 104211780B CN 201310211876 A CN201310211876 A CN 201310211876A CN 104211780 B CN104211780 B CN 104211780B
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- cancer cell
- cyclic depsipeptides
- trichoderma asperellum
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Abstract
The present invention relates to a kind of cyclic depsipeptides compound and its production and use.This compound has inhibition cancer cell proliferation activity.Cyclic depsipeptides compound shown in research belongs to bioactive peptide compound, and is constituted asymmetric cyclodepsipeptide by four lactic acid residues and four N-methyl leucine residues.By fermented and cultured trichoderma asperellum (Trichoderma asperellum), obtain mycelia interior metabolism product crude extract, the most isolated and purified go out this compound.Having not yet to see chemical constitution and the report of cancer cell multiplication inhibitory activity of this compound, be experimentally verified that, this compound can be as cancer cell multiplication inhibitor or antitumor agent.
Description
Technical field
The present invention relates to a kind of cyclic depsipeptides compound and its production and use.
Background technology
Cyclic depsipeptides compound (Cyclodepsipeptides) is the important branch of depsipeptides compound, and be often in the news tool
Have antitumor, antibacterial, antimycotic, antiviral, anti parasitic isoreactivity, their architectural feature be have one or more
Ester bond.The present inventor's research is learnt, trichoderma asperellum (Trichoderma asperellum) IBPT-2 (on October 30th, 2012
Being deposited in Wuhan University's China typical culture collection center, deposit number is: CCTCC NO:M 2012438) generation in mycelium
The crude extract thanking to product has preferable cancer cell multiplication inhibitory activity, then studies its active component.Shown in research finds
Cyclic depsipeptides compound has antitumor activity, has not yet to see chemical constitution and the cancer cell multiplication inhibitory activity of this compound
Report, therefore also there is not yet medicine related to this on market.
Summary of the invention
It is an object of the invention to provide a kind of cyclic depsipeptides compound and its production and use, this compound has
Inhibition cancer cell proliferation activity.Its structural formula is:
。
Its architectural feature is: containing four lactic acid residues and four N-methyl leucine residues, constitute asymmetrical ring
Depsipeptides compound.
Present invention also offers the preparation method of described compound: fermented and cultured trichoderma asperellum (Trichoderma
Asperellum), obtain mycelium, after carrying out broken extraction process, obtain mycelia interior metabolism product crude extract, then from slightly
In extract isolated and purified go out this compound.
Described trichoderma asperellum (Trichoderma asperellum) is trichoderma asperellum (Trichoderma
Asperellum) IBPT-2, is deposited in China typical culture collection center on October 30th, 2012, address: China
Wuhan Wuhan University, deposit number is: CCTCC NO:M 2012438.
The present invention also protects described compound purposes in preparing cancer cell multiplication inhibitor.Described cancer cell is excellent
Elect human liver cancer cell PLC as.And the purposes that this compound is in preparing antineoplastic.
The remarkable advantage of the present invention: cyclic depsipeptides compound shown in research belongs to bioactive peptide compound, and by four
Lactic acid residues and four N-methyl leucine residues constitute asymmetric cyclodepsipeptide.Described cyclic depsipeptides compound has antitumor work
Property, have not yet to see chemical constitution and the report of cell inhibitory effect activity of this compound, therefore also there is not yet on market
Medicine related to this.
Detailed description of the invention
The chemical constitution of the compound of indication in examples below:
The fermenting and producing of this compound of embodiment 1 and separation and purification
1 fermenting and producing
Produce the fermented and cultured of bacterium: by the conventional method of cultivation microorganism, take trichoderma asperellum (Trichoderma
Asperellum) (on October 30th, 2012 is deposited in Wuhan University's China typical culture collection center, and deposit number is:
CCTCC NO:M 2012438) appropriate, it is inoculated on PDA solid slope culture medium and cultivates 4 days in 28 degrees Celsius of incubators.
Take inclined-plane and cultivate the bacterial strain of 4 days in right amount, be inoculated into equipped with 400mL nutrient solution [culture medium composition (grams per liter): sweet dew
Alcohol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0, KH2PO40.5, MgSO40.3] 1000
In mL conical flask, 28 DEG C of static gas wave refrigerator are after 30 days, it is thus achieved that mycelium and zymotic fluid.
The acquisition of 2 medicinal extract
After having fermented, with gauze by mycelium and separation of fermentative broth.Soak with the solution containing acetone with water 4:1 and dissolve bacterium
Filament cell membrane, ultrasonic wave added crushes, obtains mycelial acetone crude extract by multilayer filtered through gauze.Obtain after mycelium is broken
Acetone crude extract rotary evaporation remove acetone, until the aqueous solution of remaining crude extract.Acetic acid second is added by volume for 1:2
Ester, continuous extraction twice, it is evaporated to after merging do, obtains the ethyl acetate extract of mycelia interior metabolism product.
The separation and purification of 3 compounds
This medicinal extract is by after 100-200 mesh silica gel mixed sample, with petroleum ether: dichloromethane: methyl alcohol is eluent decompression silicon
Glue chromatographic column gradient elution.Eluent follows the tracks of (suppressing human melanoma cell strain A375) through activity, obtains active component C
(methylene chloride-methanol v/v 50:1 eluate), then with petroleum ether: dichloromethane: methanol gradient component, as eluant, eluent, enters one
Step is by pressurized silica gel column chromatography gradient elution, the active subfraction C2 obtained (methylene chloride-methanol is the eluate of 20:1)
It is that solvent carries out Sephadex LH-20 gel filtration chromatography by chloroform-methanol (v/v1:2), then through RP-18 reverse phase silica gel post
Chromatograph with methanol-water (v/v 1:1) be solvent wash-out, finally by semi-preparative liquid chromatography (1010 types ODS-A, 10 × 250
Mm, 5 μm): separating flow velocity is 5 mL.min-1, flowing is 55% acetonitrile mutually, obtains shown reactive compound (94.6 mg, tR
15.43 min).
Compound as colourless crystal, cation HR-ESI-MS m/z:819.4736 [M+Na]+, molecular formula
C40H68N4O12; 1H and13The NMR data such as C-NMR are shown in Table 1.Cation second order ms fragment ion (+) ESIMS/MS m/z:
819 [M+Na]+, 692,620,493,366,294,222,95.
Table 1 compound1H and13C-NMR data (500 and 125MHz, in DMSO-d6) 1)
1) this table signals assignment is based on DEPT, HMQC and HMQC spectrum analysis result.The multiplicity of carbon signal utilizes DEPT
Method determines.
2) numeral in this hurdle and code name represent respectively1H-1H COSY spectrum in in corresponding line1It is relevant that H provides coupling
Signal1H core.
3) numeral in this hurdle and code name represent respectively HMBC compose in in corresponding line1H provides coupling coherent signal
's13C core.
4) HL in this hurdle represents that lactic acid residues, N-Me-Leu represent N-methyl leucine residue.
The test of embodiment 2 anti tumor activity in vitro
1 laboratory sample and experimental technique
The preparation of sample solution: test sample is the pure compounds of separation and purification in above-mentioned enforcement 1.Precision weighs
Appropriate amount of sample, adds 50 μ L DMSO, and ultraviolet irradiation, after one hour, adds the cell culture fluid that 950 μ L contain 10% FBS,
Arrive 1 mg.mL containing 5% DMSO-1Sample stoste.Be diluted with cell culture fluid, respectively obtain concentration be 0.1,
0.05、0.02、0.01 mg.mL-1Sample.
The squamous subculture of clone and cell uses human liver cancer cell PLC clone.The cell RPMI-containing 10% FBS
1640 culture mediums, at 37 DEG C of squamous subculture in the incubator being passed through 5% carbon dioxide.
Cell inhibitory effect activity test method
Take the tumour cell being in exponential phase, dilute with the nutrient solution containing 10% FBS, cell density is adjusted to every milli
Rise 2 × 105Cell suspension.It is paved with PBS in 96 orifice plate surroundings, then cell suspension is inoculated in 96 by every hole 100 μ L
In orifice plate, stay two to be not added with cell suspension, only add the cell culture fluid blank well as administration group Yu control group.To inoculate
96 orifice plates put into 37C, 5% CO2Incubator in cultivate 24 h.After cell attachment, sucking supernatant, the addition of every hole dilutes
Sample 100 μ L, the culture medium arranging 5% DMSO solution continues to cultivate 24 h as a control group.Carefully suck supernatant, often
Hole adds 1 mg.mL-1MTT solution 100 μ L, cultivate after 4 hours for 37 DEG C, per minute 2000 leave the heart 8 minutes, little with liquid-transfering gun
The heart sucks supernatant.Every hole adds DMSO 100 μ L, and 37 DEG C of shaking table vibrations, after being completely dissolved to crystallization, ELIASA measures 570
Every hole OD value is surveyed at nm.Take four hole mean OD value, by cell proliferation inhibition rate IR (%)=(OD compares-OD sample)/OD comparison ×
100% calculates, and calculates sample half inhibiting rate IC according to bliss method50。
2. experimental result
Cell inhibitory effect active testing result
In mtt assay is tested, this compound Proliferation Ability result to human liver cancer cell PLC cell line: IC50=24.9μM。
3. conclusion
Compound has obvious Cytostatic to tumor cell effect, can be as preparing cancer cell multiplication inhibitor or anti-swollen
Knurl agent is used for antineoplastic research.
Claims (3)
1. compound
。
The compound the most according to claim 1 purposes in preparing human liver cancer cell PLC antiblastic.
The compound the most according to claim 1 application in preparing anti-human liver cancer cell PLC medicine.
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| CN201310211876.6A CN104211780B (en) | 2013-05-30 | 2013-05-30 | A kind of cyclic depsipeptides compound and its production and use |
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| CN104211780A CN104211780A (en) | 2014-12-17 |
| CN104211780B true CN104211780B (en) | 2016-09-07 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695334B (en) * | 2014-11-25 | 2019-06-14 | 甘孜藏族自治州畜牧业科学研究所 | A kind of new trichoderma asperellum and application thereof |
| CN108570016B (en) * | 2017-03-10 | 2021-11-26 | 上海医药工业研究院 | PF1022A separation and purification method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012559A (en) * | 2012-12-21 | 2013-04-03 | 福州大学 | Peptaibol compound and application thereof |
| CN103030683A (en) * | 2012-12-21 | 2013-04-10 | 福州大学 | Peptaibol and preparation method and use thereof |
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2013
- 2013-05-30 CN CN201310211876.6A patent/CN104211780B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012559A (en) * | 2012-12-21 | 2013-04-03 | 福州大学 | Peptaibol compound and application thereof |
| CN103030683A (en) * | 2012-12-21 | 2013-04-10 | 福州大学 | Peptaibol and preparation method and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| Asperelines A-F, Peptaibols from the Marine-Derived Fungus Trichoderma asperellum;Ren J.等;《J. Nat. Prod.》;20091231(第72期);第1036-1044页 * |
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