Summary of the invention
The object of the present invention is to provide a kind of cyclic depsipeptides compound and its production and use, this compound has anticancer proliferation activity.Its structural formula is:
。
Its constitutional features is: containing four lactic acid residues and four n-formyl sarcolysine base leucine residues, constitute asymmetrical cyclic depsipeptides compound.
Present invention also offers the preparation method of described compound: fermentation culture trichoderma asperellum (
trichoderma asperellum), obtain mycelium, after carrying out broken extraction treatment, obtain mycelia interior metabolism product crude extract, then from crude extract, separation and purification goes out this compound.
Described trichoderma asperellum (
trichoderma asperellum) for trichoderma asperellum (
trichoderma asperellum) IBPT-2, be deposited in China typical culture collection center, address on October 30th, 2012: Wuhan, China Wuhan University, deposit number is: CCTCC NO:M 2012438.
The present invention also protects described compound and is preparing the purposes in cancer cell multiplication inhibitor.Described cancer cells is preferably human liver cancer cell PLC.And this compound is preparing the purposes in antitumor drug.
remarkable advantage of the present invention: shown in research, cyclic depsipeptides compound belongs to bioactive peptide compound, and forms asymmetric cyclodepsipeptide by four lactic acid residues and four n-formyl sarcolysine base leucine residues.Described cyclic depsipeptides compound has anti-tumor activity, has not yet to see the chemical structure of this compound and the report of cell inhibitory effect activity, therefore market also there is not yet medicine related to this.
Embodiment
The chemical structure of the compound of indication in following embodiment:
The fermentative production of this compound of embodiment 1 and separation and purification
1 fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, get 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, be inoculated on PDA solid slant culture base and cultivate 4 days in 28 degrees Celsius of incubators.
Get the slant culture bacterial strain of 4 days appropriate, be inoculated into and 400mL nutrient solution [substratum composition (grams per liter): N.F,USP MANNITOL 20.0, yeast extract paste 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0, KH is housed
2pO
40.5, MgSO
40.3], in 1000 mL Erlenmeyer flasks, 28 DEG C of static gas wave refrigerator, after 30 days, obtain mycelium and fermented liquid.
The acquisition of 2 medicinal extract
After having fermented, with gauze by mycelium and separation of fermentative broth.Dissolve mycelial cell wall by the solution soaking containing acetone and water 4:1, ultrasonic wave added is broken, obtains mycelial acetone crude extract by multilayer filtered through gauze.The acetone crude extract rotary evaporation removing acetone obtained after mycelium fragmentation, until the aqueous solution of remaining crude extract.By volume for 1:2 adds ethyl acetate, continuous extraction twice, is evaporated to dry after merging, obtain the ethyl acetate extract of mycelia interior metabolism product.
The separation and purification of 3 compounds
After this medicinal extract passes through 100-200 order silica gel mixed sample, with sherwood oil: methylene dichloride: methyl alcohol is elutriant decompression silica gel chromatographic column gradient elution.Elutriant follows the tracks of (suppressing human melanoma cell strain A375) through active, obtain active ingredient C (methylene chloride-methanol v/v 50:1 eluate), then with sherwood oil: methylene dichloride: methanol gradient component is eluent, further by pressurized silica gel column chromatography gradient elution, the active subfraction C2 (methylene chloride-methanol is the eluate of 20:1) obtained by chloroform-methanol (v/v1:2) for solvent carries out Sephadex LH-20 gel filtration chromatography, again through RP-18 reversed-phase silica gel column chromatography with methanol-water (v/v 1:1) for solvent elution, finally by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μm): being separated flow velocity is 5 mL.min
-1, moving phase is 55% acetonitrile, obtain shown active compound (94.6 mg,
t r15.43 min).
Compound as colourless crystal, positive ion HR-ESI-MS
m/z: 819.4736 [M+Na]
+, molecular formula C
40h
68n
4o
12;
1h and
13the NMR data such as C-NMR are in table 1.Positive ion second order ms fragmention (+) ESIMS/MS
m/z: 819 [M+Na]
+, 692,620,493,366,294,222,95.
Table 1 compound
1h and
13c-NMR data (500 and 125MHz, in DMSO-
d 6)
1)
1) this table signals assignment is based on DEPT, HMQC and HMQC spectrum analysis result.The multiplicity of carbon signal utilizes DEPT method to determine.
2) numeral in this hurdle and code name represent respectively
1h-
1with corresponding line during H COSY composes
1h provides coupling coherent signal
1h core.
3) numeral in this hurdle and code name represent in HMBC spectrum respectively with corresponding line
1h provides coupling coherent signal
13c core.
4) HL in this hurdle represents lactic acid residues, and N-Me-Leu represents n-formyl sarcolysine base 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 takes appropriate amount of sample, adds 50
μl DMSO, uv irradiating, after one hour, adds 950
μl contains the cell culture fluid of 10% FBS, obtains 1 mg.mL containing 5% DMSO
-1sample stoste.Dilute with cell culture fluid, obtaining concentration is respectively 0.1,0.05,0.02,0.01 mg.mL
-1sample.
The succeeding transfer culture of clone and cell adopts human liver cancer cell PLC clone.The RPMI-1640 substratum of cell containing 10% FBS, at 37 DEG C of succeeding transfer culture in the incubator passing into 5% carbonic acid gas.
Cell inhibitory effect activity test method
Get the tumour cell being in logarithmic phase, dilute with the nutrient solution containing 10% FBS, cell density is adjusted to every milliliter 2 × 10
5cell suspension.PBS damping fluid is paved with in 96 orifice plate surroundings, then cell suspension by every hole 100
μl is inoculated in 96 orifice plates, stays two not add cell suspension, only adds the blank well of cell culture fluid as administration group and control group.96 orifice plates inoculated are put into 37C, 5% CO
2incubator in cultivate 24 h.After cell attachment, suck supernatant, every hole adds the sample 100 diluted
μl, the substratum arranging 5% DMSO solution continues cultivation 24 h as a control group.Carefully suck supernatant liquor, every hole adds 1 mg.mL
-1mTT solution 100
μl, cultivate after 4 hours for 37 DEG C, per minute 2000 leaves the heart 8 minutes, carefully sucks supernatant liquor with liquid-transfering gun.Every hole adds DMSO 100
μl, 37 DEG C of shaking table vibrations, after dissolving completely to crystallization, microplate reader measures 570 nm places and surveys every hole OD value.Get four hole mean OD value, contrast × 100% calculate, according to bliss method calculation sample half inhibiting rate IC by cell proliferation inhibition rate IR (%)=(OD contrasts-OD sample)/OD
50.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, this compound is to the Proliferation Ability result of human liver cancer cell PLC cell strain: IC
50=24.9 μMs.
3. conclusion
Compound has obvious Cytostatic to tumor cell effect, can be used as and prepares cancer cell multiplication inhibitor or antineoplastic agent for antineoplastic research.