CN102429895B - Application of natural compound P48 in inhibition of tumor cell reproduction and growth - Google Patents
Application of natural compound P48 in inhibition of tumor cell reproduction and growth Download PDFInfo
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- CN102429895B CN102429895B CN 201110294300 CN201110294300A CN102429895B CN 102429895 B CN102429895 B CN 102429895B CN 201110294300 CN201110294300 CN 201110294300 CN 201110294300 A CN201110294300 A CN 201110294300A CN 102429895 B CN102429895 B CN 102429895B
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Abstract
The invention discloses application of a natural compound P48 in inhibition of tumor cell reproduction and growth. A structural formula of the natural compound P48 is shown in the formula I. The natural compound P48 or its pharmaceutically acceptable salt can be utilized for preparation of a drug for inhibiting eukaryote tumor cell reproduction, or a drug for prevention and/or treatment on tumors. A result of an in-vitro anticancer test shows that the natural compound P48 has good inhibition effects on breast cancer cells (MCF-7), lung cancer cells (A549), ovarian cancer cells (SKOV3), colorectal cancer cells (Caco2, SW480) and African green monkey kidney fibroblast (COS-7).
Description
Technical field
The present invention relates to a kind of native compound P48 uses in suppressing the tumor cell reproductive growth.
Background technology
Human beings'health in the tumor serious threat, and the tumor incidence of China and fatality rate are all higher.The research and development of antitumor drug at present relatively lag behind, and a kind of medicine of thorough healing tumor also do not occur.Though China develops some antitumor drug based on the external medicine of import.Therefore, develop the new type antineoplastic medicine with China's independent intellectual property right, have very important significance for the living standard that improves China people.
The special living resources that form under special habitats or extreme environment have specific genes and expression product thereof, are human precious genetic resources treasure-houses.As the cancer-resisting substance paclitaxel of bringing into play huge medical function in recent years, treatment cardiovascular diseases lovastatin, anti-malaria medicaments arteannuin etc.The development and use of functional activity material in special habitats fungus and the plant origin can produce abundant independent intellectual property right achievement, are breeding huge industrial prospect and economic benefit simultaneously.
Summary of the invention
The purpose of this invention is to provide the application of a kind of native compound P48.
Native compound P48 provided by the present invention, its structural formula is suc as formula shown in the I.
A purposes of Compound P 48 provided by the present invention is: the application in preparation inhibition eukaryote tumor cell proliferation medicine of Compound P 48 or its pharmaceutically acceptable salt.
Eukaryote described in the present invention is mammal.
Described tumor cell is cancerous cell; Described cancerous cell specifically can be lung carcinoma cell, kidney fibroblast, breast cancer cell, colon cancer cell or ovarian cancer cell.
Another purposes of Compound P 48 provided by the present invention is: acceptable salt prevents and/or treats application in the tumour medicine in preparation on Compound P 48 or its materia medica.
Described tumor is cancer; Described cancer specifically can be pulmonary carcinoma, breast carcinoma, ovarian cancer or colon cancer.
The present invention also protects a kind of medicine that suppresses the eukaryote tumor cell proliferation, and its effective ingredient is Compound P 48.
Be the medicine that prevents and/or treats tumor of effective ingredient preparation with Compound P 48 or its pharmaceutically acceptable salt, also belong to protection scope of the present invention.
The described tumour medicine that prevents and/or treats can import body such as muscle, Intradermal, subcutaneous, vein, mucosal tissue by the method for injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediation; Or mixed by other materials or wrap up the back and import body.
Be the antitumor drug of active component preparation with Compound P 48 or its pharmaceutically acceptable salt, when needing, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc.
Preventing and/or treating tumour medicine and can make various ways such as injection, tablet, powder, granule, capsule, oral liquid, unguentum, cream with the preparation of Compound P 48 or its pharmaceutically acceptable salt.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
Compound P 48 provided by the present invention is separated from the microbial solid fermentation extract and is obtained.Use modern chromatograph again, technology such as mass spectrum and NMR are carried out purification and structure elucidation to this chemical compound.
The production bacterial strain of Compound P 48 is Fructus Fici plan dish stey (Pestalotiopsis fici) N56, this strains separation is on the tea branch in area, Hangzhou, and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on the 24th in December in 2008 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC 2826.
Utilize bacterial strain Pestalotiopsis fici fermentation to prepare the method for Compound P 48, mainly may further comprise the steps:
A) Pestalotiopsis fici is carried out solid fermentation, obtain fermentation culture medium;
B) in described fermentation culture product, add organic solvent extraction, obtain extracting solution, with described extracting solution distilling under reduced pressure, obtain crude extract;
C) described crude extract is reduced pressure silica gel column chromatography and gel chromatography separation and obtain active component solution, described active component solution is separated the chemical compound that obtains formula (I) with high performance liquid chromatography.
The present invention is by making up the cell screening model to identify whether this chemical compound has the effect that suppresses tumor development.The cell screening model that makes up has breast cancer cell (MCF-7), lung carcinoma cell (A549), ovarian cancer cell (SKOV3), colon cancer cell (Caco2, SW480) etc., adopt this chemical compound of body outer cell proliferation experiment mtt assay high flux screening can suppress the reproductive growth of which kind of tumor cell, find that through test this chemical compound can suppress the reproductive growth process of above-mentioned tumor cell preferably, and this chemical compound is presented dosage effect namely along with increasing the inhibition of the reproductive growth of tumor cell of compound concentration is more good.
Description of drawings
Fig. 1 is that tumor cell is to the dosage effect of native compound P48; Breast cancer cell (MCF-7), lung carcinoma cell (A549), ovarian cancer cell (SKOV3), colon cancer cell (Caco2, SW480), African green monkey kidney fibroblast (COS-7).
Fig. 2 is the IC50 value of the tumor cell of native compound P48.
The specific embodiment
The present invention will be described below by specific embodiment, but the present invention is not limited thereto.
Experimental technique described in the following embodiment if no special instructions, is conventional method; Described reagent and material if no special instructions, all can obtain from commercial channels.
Used bacterial strain Fructus Fici plan dish stey (Pestalotiopsis fici) N56 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) in December in 2008 24 days among the embodiment, and deposit number is 2826.
The preparation of embodiment 1, native compound P48 and structure are identified
A. produce the activation of bacterial strain Fructus Fici plan dish stey (Pestalotiopsis fici) N56:
The PDA culture medium: Rhizoma Solani tuber osi 200g, glucose 20g, agar 15g, water 1000mL makes the test tube slant, and the picking mycelium is inoculated on the test tube slant, cultivates 7 days for 25 ℃;
B. produce the solid fermentation of bacterial strain Pestalotiopsis fici:
The preparation of rice medium (100g rice and 100mL water are added in the 500mL triangular flask, soaked overnight, the sterilization cooling is stand-by);
(spore concentration is 1 * 10 with the bacteria suspension for preparing
6Individual/mL) 5mL is inoculated on the rice medium, cultivated 40 days for 25 ℃;
C. treat that fermentation finishes, ethyl acetate added in the triangular flask extract three times that the evaporated under reduced pressure organic solvent obtains crude extract.
D. crude extract is used petroleum ether-ethyl acetate system (petroleum ether: ethyl acetate (v/v)=9: 1; 4: 1; 7: 3; 3: 2; 1: 1; 2: 3; 3: 7; 1: 4; 1: 9, each gradient was washed 3 times of column volumes) silica gel column chromatography that reduces pressure (8 * 30cm, tlc silica gel);
E. follow the tracks of under (to the inhibition activity of pulmonary carcinoma, breast carcinoma, ovarian cancer, colon cancer) guidance in activity, with active component (petroleum ether: ethyl acetate=7: 3) by gel chromatography separation (the gel column filler is Sephadex LH-20), mobile phase is dichloromethane: methanol=1: 1 (v/v), gained fraction (40mg) are further by reversed-phase HPLC (KramosilC18 post; 10 μ m; 10 * 250mm; 2mL/min; Be increased to 80% (linear gradient elution) behind the 70% methanol 20min and prepare reactive compound P44 (5mg; Retention time t
R=18.1min).
The P48 of method for preparing is carried out high resolution mass spectrum (HRESIMS), infrared spectrum (IR) and nmr analysis, and wherein high resolution mass spectrum provides test by Nanjing University mass spectrometric measurement center (the test instrunment model is BrukerEsquire 3000
Plus), infrared spectrum provides test (the test instrunment model is Nicolet Magna-IR 750) by chemistry institute of Peking University, and nuclear magnetic resoance spectrum provides test (the test instrunment model is Varian Mercury-400) by institute of Materia Medica,Chinese Academy of Medical Sciences.The physicochemical data of Compound P 48 sees Table 1, the hydrogen spectrum (
1H-NMR) and carbon spectrum (
13C-NMR) data see Table 2.
The physico-chemical constant of table 1. Compound P 48
The hydrogen spectrum of table 2. Compound P 48 and carbon spectrum data (Acetone-d
6)
aAt 400MH with Acetone-d
6Be the solvent test.
bAt 100MHz with Acetone-d
6Be the solvent test.
Confirmation obtains Compound P 48 and is " Pestalofones A-E, bioactive cyclohexanone derivatives from the plant endophytic fungus Pestalotiopsis fici.Bioorganic ﹠amp; Medicinal Chemistry, 2009,17,606-613 " the Compound P estalofone C that reports in the document, its structure is as follows:
The influence of embodiment 2,48 pairs of tumor cell reproductive growths of high flux screening of natural Compound P
The cell screening model that makes up: breast cancer cell (MCF-7), lung carcinoma cell (A549), ovarian cancer cell (SKOV3), colon cancer cell (Caco2, SW480), African green monkey kidney fibroblast (COS-7).
Adopt the cell proliferation experiment mtt assay to check this chemical compound to the influence of above several tumor cell reproductive growths, at first cell is inoculated into 96 orifice plates, the inoculum density difference of every kind of tumor cell, the speed of growth density of tumor cell inoculation are faster hanged down as breast cancer cell (MCF-7), ovarian cancer cell (SKOV3).According to native compound P48 the difference in the processing time of various tumor cells is also wanted the inoculum density of suitable adjustment tumor cell in addition, lower than the inoculum density of bearing tumor cell as the processing time, in order to verify that several tumor cells are to the dosage effect of this chemical compound, every kind of chemical compound selects three different chemical compound density to handle tumor cell at least, and the gradient between two concentration for the treatment of preferably is consistent, and every kind of concentration for the treatment of will arrange three repetitions.
Compound P 48 usefulness DMSO are made solvent be mixed with the solution to be measured that concentration is 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M and 0.0001 μ M respectively.
Specific as follows:
1, with cell suspension inoculation to 96 orifice plate of breast cancer cell (MCF-7), 200 μ l (5000 cells/well) are inoculated in every hole, in 37 ℃, and 5%CO
2Incubator in cultivate 24h, each hole culture fluid then inclines.The solution (concentration is respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M) of Compound P 47 is joined respectively in the culture hole of MCF-7 cell in 96 orifice plates, and each concentration is inoculated 3 holes, and every hole adds 200 μ l; Be placed on 37 ℃, 5%CO
2Incubator in cultivate 72h.
2,200 μ l (4000 cells/well) are inoculated in the every hole of ovarian cancer cell (SKOV3); The concentration of Compound P 47 solution that add is respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M; The processing time of the SKOV3 of P47 is 72h, and all the other are with 1.
3,200 μ l (7500 cells/well) are inoculated in the every hole of colon cancer cell (Caco2); The concentration of Compound P 47 solution that add is respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M; The processing time of the Caco2 of P47 is 72h, and all the other are with 1.
4,200 μ l (7500 cells/well) are inoculated in the every hole of colon cancer cell (SW480); The concentration of Compound P 47 solution that add is respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M; The processing time of the SW480 of P47 is 72h, and all the other are with 1.
5,200 μ l (10000 cells/well) are inoculated in the every hole of African green monkey kidney fibroblast (COS-7); The concentration of Compound P 47 solution that add is respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M; The processing time of the COS-7 of P47 is 72h, and all the other are with 1.
6,200 μ l (10000 cells/well) are inoculated in the every hole of lung carcinoma cell (A549); The concentration of Compound P 47 solution that add is respectively 10 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, 0.001 μ M, 0.0001 μ M; The processing time of the A549 of P47 is 72h, and all the other are with 1.
Four hours every holes add 20ul MTT processing tumor cell before measuring the MTT value, wash the tumor cell culture base after four hours off, every hole adds the DMSO of 150ul, concussion was measured MTT value (optical density OD value) under the condition of microplate reader 490-630nm dual wavelength in about ten minutes again under the low rate then, by formula calculated the growth of tumour cell suppression ratio.
OD in this experiment
Contrast, OD
ExperimentValue is deduction and falls OD
BlankValue.
48 pairs of above-mentioned cyto-inhibitions of Compound P the results are shown in Figure 1.
Embodiment 3, mensuration native compound P48 are to the IC50 value of several tumor cells
Measure the IC50 value of the breast cancer cell of native compound P48 (MCF-7), lung carcinoma cell (A549), ovarian cancer cell (SKOV3), colon cancer cell (Caco2, SW480), African green monkey kidney fibroblast (COS-7) etc., at first to determine six concentration for the treatment of of native compound P48, be consistent between each Concentraton gradient, six concentration are respectively: 0.0001,0.001,0.01,0.1,1.0,10 (uM), every kind of concentration is handled three repetitions will be set.Contrast is handled with DMSO.Adjust tumor cell at the inoculum density (concrete inoculum density and processing time are with embodiment 2) of 96 orifice plates according to the reproductive growth speed of processing time of chemical compound and tumor cell.Tumor cell is handled with 20ulMTT in four hours every holes before measuring the MTT value, wash the tumor cell culture base after four hours off, every hole adds the DMSO of 150ul, and concussion about ten minutes is measured the MTT value under the condition of microplate reader 490-630nm dual wavelength again under the low rate then.According to the MTT value of each compound treatment concentration that obtains, thereby adopt the computing formula of IC50 value to obtain chemical compound to the IC50 value of tumor cell.The results are shown in Figure 2.
Calculate IC
50Value computing formula: lgIC50=Xm-I (P-(3-Pm-Pn)/4)
The Xm:lg maximal dose
I:lg (maximal dose/face mutually dosage)
P: positive reaction rate sum
Pm: maximum positive reaction rate
Pn: minimum positive reaction rate
As can be seen from Figure 2 the proliferation inhibiting effect of 48 couples of COS-7 of Compound P and SW480 is best, and IC50 is 0.22uM (0.114ug/ml); Next is lung cell A549, its IC50=0.29uM (0.15ug/ml); Be MCF-7 then, IC50=0.59uM (0.307ug/ml); Then be Caco2, IC50=0.93uM (0.483ug/ml); Be SKOV3 at last, IC50=2.07uM (1.08ug/ml).
Claims (2)
1. the chemical compound shown in the formula I or its pharmaceutically acceptable salt application in preparation inhibition eukaryote tumor cell proliferation medicine;
(formula I)
Wherein, described eukaryote is mammal; Described tumor cell is cancerous cell; Described cancerous cell is lung carcinoma cell, kidney fibroblast, breast cancer cell or ovarian cancer cell; Described kidney fibroblast is African green monkey kidney fibroblast COS-7, and described lung carcinoma cell is lung cell A549, and described breast cancer cell is breast cancer cell MCF-7, and described ovarian cancer cell is SKOV3.
2. acceptable salt prevents and/or treats application in the tumour medicine in preparation on the chemical compound shown in the formula I described in the claim 1 or its materia medica; Wherein, described tumor is cancer; Described cancer is pulmonary carcinoma, ovarian cancer or breast carcinoma.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538537A (en) * | 2009-02-13 | 2009-09-23 | 中国科学院微生物研究所 | Pestalotiopsis fici and discovery and application of generated anti-tumor active compounds thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101538537A (en) * | 2009-02-13 | 2009-09-23 | 中国科学院微生物研究所 | Pestalotiopsis fici and discovery and application of generated anti-tumor active compounds thereof |
Non-Patent Citations (3)
| Title |
|---|
| Ling Liu, et al..pestalofones A-E, bioactive cyclohexanone derivatives from the plant endophytic fungus pestalotiopsis fici.《bioorganic & * |
| LingLiu et al..pestalofones A-E |
| medicinal chemistry》.2009,第17卷第606-613页. * |
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