CN103265522B - Lactone derivative derived from trichoderma citrinoviride and application thereof - Google Patents
Lactone derivative derived from trichoderma citrinoviride and application thereof Download PDFInfo
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- CN103265522B CN103265522B CN201310208563.5A CN201310208563A CN103265522B CN 103265522 B CN103265522 B CN 103265522B CN 201310208563 A CN201310208563 A CN 201310208563A CN 103265522 B CN103265522 B CN 103265522B
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- lactone
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Abstract
The invention relates to a lactone derivative derived from trichoderma citrinoviride and application thereof. The compound contains a molecular skeleton of hetero-oxygen atom pentacyclic lactone, and two methyls on the ring are located at the same side of the molecule. Verified by experiments, the lactone derivative has excellent anti-tumor activity. Besides, the lactone derivative can be applied to preparation of a cell proliferation inhibition drug or an anti-tumor drug and used for anti-tumor research.
Description
Technical field
The present invention relates to a kind of lactone derivatives and application thereof that comes from tangerine green trichoderma.
Background technology
In recent decades, in research and development novel anti-tumor medicine process, many natural lactones of antitumour activity that have have been found.There is the natural lactone of antitumour activity and mostly derive from marine organisms, its structure uniqueness, mechanism of action novelty, not only can provide lead compound, and for finding that new action target spot provides foundation.In addition, study in recent years more Iejimalide class, anterior canal algae lactone, occupied tongue discodermolide compounds also for finding new antitumor action target spot, the anticarcinogen of exploitation novel mechanism has been brought into play important effect, becomes the instrument of research new antitumoral medicine.
The inventor studies and learns, tangerine green trichoderma (
trichoderma citrinoviride.) IBPT-4 (has been deposited in Chinese Typical Representative culture collection center on January 25th, 2013, address: Wuhan, China Wuhan University, deposit number is: CCTCC NO:M 2013055) the crude extract of tunning have good tumor cell proliferation inhibition activity, then its activeconstituents is studied.Research finds that wherein lactone compound has anti-tumor activity, has not yet to see the chemical structure of this compound and the report of tumor cell proliferation inhibition activity, therefore on market, also there is not yet medicine related to this.
Summary of the invention
The object of the present invention is to provide a kind of lactone derivatives and application thereof that comes from tangerine green trichoderma.
The present invention first relate to a strain tangerine green trichoderma (
trichoderma citrinoviride.) IBPT-4, this bacterial strain has been deposited in Chinese Typical Representative culture collection center, address on January 25th, 2013: Wuhan, China Wuhan University, deposit number is: CCTCC NO:M 2013055.
The purposes of described bacterial strain is, by tangerine green trichoderma (
trichoderma citrinoviride.) IBPT-4 carries out fermentation culture, mycelium and fermentation broth extract are isolated to the compound with tumor cell proliferation inhibition activity.
This structural formula of compound is:
。
Its constitutional features is: the molecular skeleton that contains an assorted Sauerstoffatom five rings lactone, encircle two methyl in molecule homonymy.
The present invention has also protected described compound in the purposes of preparing in inhibition tumor cell propagation medicine, and this compound is in the purposes of preparing in antitumor drug.
Remarkable advantage of the present invention: the molecular structure that contains an assorted Sauerstoffatom five rings lactone shown in research is extremely rare, described lactone derivatives has significant anti-tumor activity, have not yet to see the chemical structure of this compound and the report of tumor cell proliferation inhibition activity, therefore on market, also there is not yet medicine related to this.
Embodiment
The chemical structure of the compound of indication in following embodiment:
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, get tangerine green trichoderma (
trichoderma citrinoviride.) IBPT-4 (has been deposited in Chinese Typical Representative culture collection center on January 25th, 2013, address: Wuhan, China Wuhan University, deposit number is: CCTCC NO:M 2013055) appropriate, be inoculated on PDA solid slant culture base and cultivate 4 days in 28 ℃ of incubators.
Get the slant culture tangerine green trichoderma of 4 days (
trichoderma citrinoviride.) IBPT-4 is appropriate, be inoculated into 400mL nutrient solution [nutrient solution 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, NaCL, 6.0 constant volumes] 1000mL Erlenmeyer flask in, 28 ℃ of static cultivations are after 30 days, obtain mycelium and fermented liquid.
The acquisition of 2 medicinal extract
With gauze by mycelium and separation of fermentative broth.By fermented liquid and ethyl acetate 1:2(v/v) extracting twice, extraction liquid underpressure distillation, to dry, obtains the ethyl acetate extract of fermented liquid.Mycelium is with containing the aqueous solution ultrasonication of 70%-80% acetone 3 times, and after removing residue clear liquid being merged, concentrating under reduced pressure is removed acetone, by volume for 1:2 adds ethyl acetate extracting twice, is evaporated to dry mycelium medicinal extract.To after mycelium and the merging of fermented liquid medicinal extract, obtain the total medicinal extract of extract.
The separation and purification of 3 compounds
This medicinal extract is by after 100-200 order silica gel mixed sample, take sherwood oil: methylene dichloride: methyl alcohol is as decompression silica gel chromatographic column gradient elution for elutriant.Elutriant is through active tracking (human melanoma cell strain A375 is suppressed), obtain active component A (methylene dichloride eluate), then take sherwood oil: methylene dichloride: methyl alcohol gradient composition is eluent, further by pressurized silica gel column chromatography gradient elution, the active subfraction A2 (eluate that methylene chloride-methanol is 20:1) obtaining carries out Sephadex LH-20 gel filtration chromatography by chloroform-methanol (v/v1:2) for solvent, remove impurity through reverse phase silica gel chromatographic column (60%MeOH), finally by half preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μ are m): separating flow velocity is 5 mL/min, moving phase is that 25% acetonitrile is containing 0.1% TFA, (9 mg of compound shown in obtaining,
t r11.6 min).
The unformed powder of compound, high resolution mass spectrum HRESI-MS exists
m/z283.1183 places provide molecular ion peak [M – H]
–, (Calcd for C
14h
19o
6, 283.1182), prompting molecular weight is 284, infers that in conjunction with spectral information molecular formula is C
14h
20o
6.
1h and
13the NMR data such as C-NMR are in table 1.
Table 1 compound
1h and
13c-NMR data (500 MHz, in CDCl
3)
a)
A) this table signal belongs to based on DEPT, HMQC, NOE and HMBC spectrum analysis result.The multiple degree of carbon signal utilizes DEPT method to determine.
B) numeral in this hurdle and code name represent respectively in HMBC spectrum with corresponding line in
1h provides coupling coherent signal
13c core.
C) numeral in this hurdle and code name represent respectively in COSY spectrum with corresponding line in
1h provides coupling coherent signal
1h core.
D) numeral in this hurdle and code name represent respectively in NOE spectrum with corresponding
1h provides coupling coherent signal
1h core.
The test of embodiment 2 anti tumor activity in vitro
1 laboratory sample and experimental technique
The preparation test sample of sample solution is the pure compounds of separation and purification in above-mentioned enforcement 1.Precision takes appropriate sample, is mixed with the solution of desired concn with methyl alcohol, active for surveying.
The succeeding transfer culture of clone and cell adopts Humanmachine tumour A375 clone, and the RPMI-1640 substratum containing 10%FBS for A375 cell, at 37 ℃ of succeeding transfer culture in the incubator that passes into 5% carbonic acid gas.
Cell inhibitory effect activity test method
The Humanmachine tumour A375 cell that tetrazolium (MTT) method is taken the logarithm vegetative period, is adjusted to every milliliter 2 × 10 by cell density
5individual cell, is inoculated in 96 porocyte culture plates by every hole 200 microlitres, passes into 5% CO in 37 ℃
2incubator in cultivate 4 hours.Every hole adds 2 microlitre sample liquid or blank solutions, cultivates after 24 hours, and every hole adds MTT liquid (every milliliter of 5 milligrams of normal saline solutions of MTT) 10 microlitres, continue cultivate 4 hours, 37 ℃, 2000 revs/min centrifugal 8 minutes, suck supernatant.Every hole adds each 100 microlitres of DMSO, vibrates 15 minutes on micro oscillator, after dissolving completely, utilizes MD company to produce SPECTRAMAX Plus type microplate reader and measures light absorption value (OD) value of every hole at 570nm place to crystallization.In same 96 orifice plates, each concentration of sample all arranges three holes, separately 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 first done corresponding acellular zeroing, then gets the three average OD values in hole by IR (%)=(OD
blank-OD
sample)/OD
blank× 100% formula is calculated cell proliferation inhibition rate under each concentration (IR%).
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, according to the A375 cell proliferation inhibition rate of this compound of different concns, application SPSS16.0 software carries out data processing calculation of half inhibitory concentration IC
50value.The results are shown in Table 2.
The inhibition activity of table 2 compound to Humanmachine tumour A375 cell proliferation
3. conclusion
Compound has obvious Cytostatic to tumor cell effect, can be used as and prepares inhibition of cell proliferation or antineoplastic agent for antineoplastic research.
Claims (3)
1. compound
.
2. compound claimed in claim 1 is in the application of preparing in inhibition tumor cell propagation medicine.
3. compound claimed in claim 1 is in the application of preparing in antitumor drug.
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| JP特开2012-105555A 2012.06.07 |
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