CN106389418B - Derived from tangerine green trichoderma mould enol E1 liver cancer application - Google Patents
Derived from tangerine green trichoderma mould enol E1 liver cancer application Download PDFInfo
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- CN106389418B CN106389418B CN201610819907.XA CN201610819907A CN106389418B CN 106389418 B CN106389418 B CN 106389418B CN 201610819907 A CN201610819907 A CN 201610819907A CN 106389418 B CN106389418 B CN 106389418B
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- liver cancer
- trichoderma
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- alkaloid compound
- green trichoderma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
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- C—CHEMISTRY; METALLURGY
- 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
- C12P17/10—Nitrogen as only ring hetero atom
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Abstract
The invention discloses a kind of alkaloid compound derived from tangerine green trichoderma liver cancer application.The structure feature of the compound is: the molecular skeleton containing five cyclic amides, connect a ten carbon saturated fat long-chains containing carbonyl, a methyl connected on N, there are two hydroxyl groups in molecule.It is verified by experiments, the alkaloid compound has preferable inhibitory activity to liver cancer cells, can be used for preparing hepatoma cell proliferation and inhibits drug or medicines resistant to liver cancer, for antitumor research.
Description
Technical field
The present invention relates to a kind of alkaloid compound mould enol E1 derived from tangerine green trichoderma liver cancer application.
Background technique
Alkaloid is a kind of organic compounds containing nitrogen generated by biological cometabolism, the alkaloid type in nature compared with
It is more, greatly mostly from plant, therefore there is the title of vegetable soda again.Alkaloid has important physiological action to humans and animals, including flat
Antibechic, hypoglycemic, reducing blood lipid, antibacterial, antitumor, analgesia etc. are breathed heavily, wherein the most prominent with antibacterial, anti-tumor activity.Natural knot
Structure alkaloid is the important sources that lead compound is found in innovation drug research, has been applied to clinical alkaloidal drug at present
Through nearly hundred kinds.The study found that some marine fungis can generate structure novel, activity good biology during cometabolism
Alkali has good medicinal and industrialization prospect.
The present inventor studies and learns, tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4(is in 2013
On January 25, in is deposited in China typical culture collection center, and address: Wuhan Wuhan University, deposit number are: CCTCC NO:
M 2013055) the crude extract of tunning have good tumor cell proliferation inhibition activity, its active constituent is carried out then
Research.The study found that shown alkaloid compound has anti-tumor activity for liver cancer, and have not yet to see the compound
Chemical structure and its report for hepatoma cell proliferation inhibitory activity, therefore in the market also there is not yet medicine related to this
Object.
Summary of the invention
The purpose of the present invention is to provide a kind of alkaloid compound derived from tangerine green trichoderma liver cancer application.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of alkaloid compound derived from tangerine green trichoderma inhibits for hepatoma cell proliferation the use in drug in preparation
On the way or the compound is preparing the purposes in medicines resistant to liver cancer；
The chemical structural formula of the compound are as follows:
；
Its structure feature is: the molecular skeleton containing five cyclic amides connects a ten carbon saturation containing a carbonyl
Connect that a methyl, there are two hydroxyl groups in molecule on fatty long-chain, N.
The compound be by tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4 progress fermented and cultured,
Then isolated from its mycelium and fermentation broth extract；The tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4 is deposited in China typical culture collection center on January 25th, 2013, address: Wuhan
Wuhan University, deposit number are: CCTCC NO:M 2013055.
Remarkable advantage of the invention is: present invention gained compound is the alkaloid of a structure novel, is had aobvious
The resisting liver cancer activity of work, and have not yet to see the chemical structure of the compound and its report for hepatoma cell proliferation inhibitory activity
Road, therefore in the market also there is not yet drug related to this.
Detailed description of the invention
Fig. 1 is the present invention gained main COSY and HMBC signal of alkaloid compound.
Specific embodiment
The chemical structural formula of signified compound in the following example are as follows:
。
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 tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4(is deposited in China typical culture collection center on January 25th, 2013, address: Wuhan
Wuhan University, deposit number are: CCTCC NO:M 2013055) in right amount, it is inoculated into PDA solid slope culture medium, at 28 DEG C
It is cultivated 4 days in incubator.
Take inclined-plane culture 4 days tangerine green trichoderma (Trichoderma citrinoviride.) appropriate IBPT-4, it is inoculated into
Equipped with 400mL culture solution, [culture solution forms (grams per liter): mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, Portugal
Grape sugar 10.0, KH2PO40.5, MgSO40.3, NaCl 6.0 constant volume] 1000mL conical flask in, 28 DEG C static gas wave refrigerator 30 days
Afterwards, mycelium and fermentation liquid are obtained.
2. the acquisition of medicinal extract
With gauze by mycelium and separation of fermentative broth.Fermentation liquid and ethyl acetate 1:2(v/v) are extracted twice, extract liquor
Vacuum distillation obtains the ethyl acetate extract of fermentation liquid to doing.Mycelium is then with the aqueous solution ultrasonication of the acetone containing 70%-80%
3 times, residue is removed, removal acetone is concentrated under reduced pressure after clear liquid is merged, 1:2 is added ethyl acetate and is extracted twice by volume, subtracts
Pressure is concentrated to dryness, and obtains mycelial ethyl acetate extract.It is mentioned after the ethyl acetate extract of mycelium and fermentation liquid is merged
Take the total medicinal extract of object.
3. the separation and purification of compound
After extract total medicinal extract 100-200 mesh silica gel mixed sample, using petroleum ether: methylene chloride: methanol is used as eluent
Depressurize silica gel chromatographic column gradient elution.Eluent obtains active component B(methylene chloride-methanol 50:1 v/v by activity tracking
Eluate), then using petroleum ether: methylene chloride: methanol is further eluted by pressurized silica gel column chromatographic grade as eluant, eluent,
Obtained active subfraction B1(methylene chloride-methanol 20:1 v/v eluate), then with chloroform-methanol (1:2 v/v) for solvent
Sephadex LH-20 gel filtration chromatography is carried out, finally by semi-preparative liquid chromatography (1010 type ODS-A, 10 × 250 mm, 5 μ
M): separation flow velocity be 5 mL/min, mobile phase be 85% acetonitrile contain 0.1% TFA, obtain shown compound (32.1 mg,t R
20.7min).
Compound: light yellow oil, high resolution mass spectrum HRESI-MS existm/z324.2161 place provides molecular ion peak
[M–H]–, (Calcd for C18H30NO4, 324.2175), prompting molecular weight is 325, speculates that molecular formula is in conjunction with spectral information
C18H31NO4。1H and13The NMR datas such as C-NMR are shown in Table 1.
1 compound of table1H and13C-NMR data (500 MHz, CDCl3)a)
The test of 2 anti tumor activity in vitro of embodiment
1. laboratory sample and experimental method
The preparation of sample solution: test sample is the pure compounds of separation and purification in embodiment 1.Precision weighs suitable
Sample is measured, the solution of required concentration is configured to methanol, for surveying activity.
The squamous subculture of cell line and cell uses liver cancer cell lines, and cell uses the RPMI-1640 containing 10% FBS to cultivate
Base, 37 DEG C, be passed through 5% CO2Incubator in squamous subculture.
Cell inhibitory effect activity test method
Tetrazolium (MTT) method: cell density is adjusted to every milliliter 2 × 10 by the cell of logarithmic growth phase5A cell,
Be inoculated in 96 porocyte culture plates for 200 microlitres by every hole, in 37 DEG C, be passed through 5% CO2Incubator in cultivate 4 hours.Every hole
2 microlitres of sample liquids or blank solution is added, after culture 24 hours, every hole adds MTT liquid (containing 5 milligrams in every milliliter of physiological saline
MTT) 10 microlitres, continue culture 4 hours, 37 DEG C, 2000 revs/min are centrifuged 8 minutes, suck supernatant.DMSO each 100 is added in every hole
Microlitre, it is vibrated 15 minutes on micro oscillator, until producing SPECTRAMAX Plus type using MD company after crystallization is completely dissolved
Microplate reader measures light absorption value (OD value) of every hole at 570nm.In 96 orifice plate of same, each concentration samples are respectively provided with three holes,
Separately set three hole blank controls and cell-free zeroing hole (if drug has color to do the cell-free zeroing of relative medicine concentration).Each hole
OD value first does corresponding cell-free zeroing, then takes three hole mean OD values, by IR(%)=(ODBlank control-ODSample)/ODBlank control× 100% meter
Calculate cell proliferation inhibition rate (IR%) under each concentration.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, according to the hepatoma cell proliferation inhibiting rate of the compound of various concentration, using SPSS16.0
Software carries out data processing and calculation of half inhibitory concentration IC50Value.It the results are shown in Table 2.
Inhibitory activity of 2 compound of table to hepatoma cell proliferation
3. conclusion
The compound have apparent hepatoma cell proliferation inhibiting effect, can be used for preparing hepatoma cell proliferation inhibitor or
Medicines resistant to liver cancer, for antitumor research.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of alkaloid compound derived from tangerine green trichoderma is preparing the purposes in hepatoma cell proliferation inhibition drug, special
Sign is: the chemical structural formula of the alkaloid compound are as follows:
；The alkaloid compound be by tangerine green trichoderma (Trichoderma citrinoviride) IBPT-4 progress fermented and cultured, it is then isolated from its fermentation liquid and hypha extract.
2. a kind of alkaloid compound derived from tangerine green trichoderma is preparing the purposes in medicines resistant to liver cancer, it is characterised in that: institute
State the chemical structural formula of alkaloid compound are as follows:
；The alkaloid compound be by tangerine green trichoderma (Trichoderma citrinoviride) IBPT-4 progress fermented and cultured, it is then isolated from its fermentation liquid and hypha extract.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610819907.XA CN106389418B (en) | 2016-09-13 | 2016-09-13 | Derived from tangerine green trichoderma mould enol E1 liver cancer application |
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| CN201610819907.XA CN106389418B (en) | 2016-09-13 | 2016-09-13 | Derived from tangerine green trichoderma mould enol E1 liver cancer application |
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| CN106389418A CN106389418A (en) | 2017-02-15 |
| CN106389418B true CN106389418B (en) | 2019-06-07 |
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| CN201610819907.XA Expired - Fee Related CN106389418B (en) | 2016-09-13 | 2016-09-13 | Derived from tangerine green trichoderma mould enol E1 liver cancer application |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110136752A1 (en) * | 2009-12-04 | 2011-06-09 | Novobiotic Pharmaceuticals Llc | Novel antibiotics |
| CN103865808A (en) * | 2014-03-10 | 2014-06-18 | 福州大学 | Novel anti-tumor application of penicillium enol A1 from penicillium citrinum |
-
2016
- 2016-09-13 CN CN201610819907.XA patent/CN106389418B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110136752A1 (en) * | 2009-12-04 | 2011-06-09 | Novobiotic Pharmaceuticals Llc | Novel antibiotics |
| CN103865808A (en) * | 2014-03-10 | 2014-06-18 | 福州大学 | Novel anti-tumor application of penicillium enol A1 from penicillium citrinum |
Non-Patent Citations (2)
| Title |
|---|
| Penicillenols from Penicillium sp. GQ-7, an Endophytic Fungus Associated with Aegiceras corniculatum;Zhen-Jian LIN等;《Chem. Pharm. Bull.》;20071112;第56卷(第2期);第217-221页，尤其是第217页左栏第2段，第220页右栏第6段，图1，表3 * |
| TUMONOIC ACIDS K AND L, NOVEL METABOLITES FROM THE MARINE-DERIVED FUNGUS PENICILLIUM CITRINUM;Li Chen等;《HETEROCYCLES》;20111208;第85卷(第2期);第413-419页，尤其是第414页图1，416页6-8行 * |
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