CN106432036B - Derived from tangerine green trichoderma mould enol E1 and prepare anti-oral cavity epidermal carcinoma medicinal application - Google Patents
Derived from tangerine green trichoderma mould enol E1 and prepare anti-oral cavity epidermal carcinoma medicinal application Download PDFInfo
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- CN106432036B CN106432036B CN201610819908.4A CN201610819908A CN106432036B CN 106432036 B CN106432036 B CN 106432036B CN 201610819908 A CN201610819908 A CN 201610819908A CN 106432036 B CN106432036 B CN 106432036B
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- China
- Prior art keywords
- oral cavity
- trichoderma
- epidermal carcinoma
- tangerine green
- green trichoderma
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D207/36—Oxygen or sulfur atoms
- C07D207/38—2-Pyrrolones
-
- 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
-
- 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
Abstract
The present invention relates to the mould enol E1 derived from tangerine green trichoderma and prepare anti-oral cavity epidermal carcinoma medicinal application.The compound structure is characterized in: the molecular skeleton containing five cyclic amides, containing connecting on one ten carbon saturated fat long-chain of a carbonyl connection, N, a methyl, there are two hydroxyl groups for molecule.It is verified by experiments, the alkaloid compound has preferable inhibitory activity to oral cavity epidermal carcinoma.Preparation oral cavity epidermal carcinoma cytostatic object or anti-oral cavity epidermis cancer drug be can be used as antitumor research.
Description
Technical field
The invention belongs to medicinal chemistry arts, and in particular to a kind of mould enol E1 derived from tangerine green trichoderma and prepare anti-mouth
Chamber epidermal carcinoma medicinal 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 has been (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, then to its active constituent
It is studied.Research finds that shown alkaloid compound has anti-tumor activity for oral cavity epidermal carcinoma, has not yet to see this
The chemical structure of compound and be directed to the active report of oral cavity epidermal carcinoma cell inhibitory effect, therefore in the market also there is not yet with
This relevant drug.
Summary of the invention
The purpose of the present invention is to provide a kind of alkaloid compound derived from tangerine green trichoderma and its in oral cavity epidermal carcinoma
The application of aspect.
Present invention firstly relates to one plant of tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4, the bacterial strain
It is deposited in China typical culture collection center on January 25th, 2013, address: Wuhan Wuhan University, deposit number are:
CCTCC NO:M 2013055.
The purposes of the bacterial strain is, by tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4 sent out
Ferment culture, mycelium and fermentation broth extract are isolated to the compound with tumor cell proliferation inhibition activity.
The structural formula of compound are as follows:
。
Its structure feature is: the molecular skeleton containing five cyclic amides connects a ten carbon saturation containing a carbonyl
On fatty long-chain, N connect a methyl, there are two hydroxyl groups for molecule.
The present invention also protects the compound in preparation for the use in oral cavity epidermal carcinoma cytostatic object
On the way and the compound is preparing the purposes in anti-oral cavity epidermis cancer drug.
Remarkable advantage of the invention: the compound shown in studying is the alkaloid of a structure novel, the alkaloids
Compound has significant anti-oral cavity epidermal carcinoma activity, has not yet to see the chemical structure of the compound and for oral cavity epidermal carcinoma
The active report of cell inhibitory effect, therefore in the market also there is not yet drug related to this.
Detailed description of the invention
The main COSY and HMBC signal of Fig. 1.
Specific embodiment
The chemical structure of signified compound in the following example:
。
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: military
Chinese Wuhan University, deposit number are: CCTCC NO:M 2013055) in right amount, it is inoculated into PDA solid slope culture medium 28
It is cultivated 4 days in DEG C 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 form (grams per liter): mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0,
Glucose 10.0, KH2PO40.5, MgSO40.3, NaCL 6.0, constant volume is into 1000mL conical flask], 28 DEG C of static gas wave refrigerators 30
After it, mycelium and fermentation liquid are obtained.
The acquisition of 2 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%
It 3 times, removes and removal acetone is concentrated under reduced pressure after residue merges clear liquid, be extracted twice with ethyl acetate is added for 1:2 by volume,
It is concentrated to dryness to obtain mycelium medicinal extract.The total medicinal extract of extract is obtained after mycelium and fermentation liquid medicinal extract are merged.
The separation and purification of 3 compounds
After the medicinal extract passes through 100-200 mesh silica gel mixed sample, using petroleum ether: methylene chloride: methanol depressurizes silicon as eluent
Glue chromatographic column gradient elution.Eluent obtains active component B (methylene chloride-methanol v/v 50:1 elution by activity tracking
Object), then with petroleum ether: methylene chloride: methanol gradient group is divided into eluant, eluent, is further washed by pressurized silica gel column chromatographic grade
It is de-, obtained active subfraction B1 (eluate that methylene chloride-methanol is 20:1) with chloroform-methanol (v/v1:2) be solvent into
Row Sephadex LH-20 gel filtration chromatography, 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 molecular formula in conjunction with spectral information
For C18H31NO4。1H and13The NMR datas such as C-NMR are shown in Table 1.
1 compound of table1H and13C-NMR data (500 MHz, in CDCl3)a)
A) this table signals assignment is based on DEPT, HMQC and HMBC spectrum analysis result.The type of carbon signal utilizes the side DEPT
Method determines.
The test of 2 anti tumor activity in vitro of embodiment
1 laboratory sample and experimental method
The preparation test sample of sample solution is the pure compounds of separation and purification in above-mentioned implementation 1.Precision weighs
Appropriate amount of sample is configured to the solution of required concentration with methanol, for surveying activity.
The squamous subculture of cell line and cell uses oral cavity epidermal carcinoma cell lines, RPMI-1640 of the cell containing 10% FBS
Culture medium, in 37 DEG C of squamous subcultures in the incubator for being passed through 5% carbon dioxide.
Cell inhibitory effect activity test method
The cell of tetrazolium (MTT) method logarithmic growth phase, is adjusted to every milliliter 2 × 10 for cell density5A cell,
It is inoculated in 96 porocyte culture plates for 200 microlitres by every hole, is passed through 5% CO in 37 DEG C2Incubator 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, and (every milliliter of 5 milligrams of physiology salts of MTT are water-soluble
Liquid) 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.Each concentration of sample is respectively provided with three in 96 orifice plate of same
Hole separately sets 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% formula calculates cell proliferation inhibition rate (IR%) under each concentration.
2. experimental result
Cell inhibitory effect active testing result
In mtt assay test, according to the oral cavity epidermal carcinoma cell proliferation inhibition rate of the compound of various concentration, application
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 oral cavity epidermal carcinoma cell Proliferation
3. conclusion
Compound has apparent oral cavity epidermal carcinoma cell inhibitory effect effect, can be used as preparation oral cavity epidermis cancer cell and increases
Inhibitor or anti-oral cavity epidermis cancer drug are grown 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. the mould enol E1 derived from tangerine green trichoderma is preparing the purposes in oral cavity epidermal carcinoma cytostatic object, penem
Its chemical structure of alcohol E1 is；By tangerine green trichoderma
(Trichoderma citrinoviride.) IBPT-4 progress fermented and cultured, then from its mycelium and fermentation broth extract
Isolated；The tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4 on January 25th, 2013 protect
China typical culture collection center is ensconced, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013055.
2. the mould enol E1 derived from tangerine green trichoderma is preparing the purposes in anti-oral cavity epidermis cancer drug；Its chemistry of mould enol E1
Structure is；By tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4 progress fermented and cultured, it is then isolated from its mycelium and fermentation broth extract；Institute
State tangerine green trichoderma (Trichoderma citrinoviride.) IBPT-4 be deposited in on January 25th, 2013 Chinese Typical Representative training
Object collection is supported, address: Wuhan Wuhan University, deposit number are: CCTCC NO:M 2013055.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610819908.4A CN106432036B (en) | 2016-09-13 | 2016-09-13 | Derived from tangerine green trichoderma mould enol E1 and prepare anti-oral cavity epidermal carcinoma medicinal application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610819908.4A CN106432036B (en) | 2016-09-13 | 2016-09-13 | Derived from tangerine green trichoderma mould enol E1 and prepare anti-oral cavity epidermal carcinoma medicinal application |
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| Publication Number | Publication Date |
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| CN106432036A CN106432036A (en) | 2017-02-22 |
| CN106432036B true CN106432036B (en) | 2019-05-10 |
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| CN201610819908.4A Expired - Fee Related CN106432036B (en) | 2016-09-13 | 2016-09-13 | Derived from tangerine green trichoderma mould enol E1 and prepare anti-oral cavity epidermal carcinoma medicinal application |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997035565A1 (en) * | 1996-03-27 | 1997-10-02 | Toray Industries, Inc. | Ketone derivatives and medicinal use thereof |
| US20110136752A1 (en) * | 2009-12-04 | 2011-06-09 | Novobiotic Pharmaceuticals Llc | Novel antibiotics |
| CN103865809A (en) * | 2014-03-10 | 2014-06-18 | 福州大学 | Novel anti-tumor application of penicillium enol B1 from penicillium citrinum |
| CN103865808A (en) * | 2014-03-10 | 2014-06-18 | 福州大学 | Novel anti-tumor application of penicillium enol A1 from penicillium citrinum |
| CN104531540A (en) * | 2014-12-18 | 2015-04-22 | 福州大学 | Antitumor application of penicillium enol A2 from penicillium citrinum |
-
2016
- 2016-09-13 CN CN201610819908.4A patent/CN106432036B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997035565A1 (en) * | 1996-03-27 | 1997-10-02 | Toray Industries, Inc. | Ketone derivatives and medicinal use thereof |
| US20110136752A1 (en) * | 2009-12-04 | 2011-06-09 | Novobiotic Pharmaceuticals Llc | Novel antibiotics |
| CN103865809A (en) * | 2014-03-10 | 2014-06-18 | 福州大学 | Novel anti-tumor application of penicillium enol B1 from penicillium citrinum |
| CN103865808A (en) * | 2014-03-10 | 2014-06-18 | 福州大学 | Novel anti-tumor application of penicillium enol A1 from penicillium citrinum |
| CN104531540A (en) * | 2014-12-18 | 2015-04-22 | 福州大学 | Antitumor application of penicillium enol A2 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》;20080229;第56卷(第2期);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页第8行、第416页第6-8行、图1 * |
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