CN102988334A - Application of 2-methoxy-6-acetyl-7-methyljuglone (MAM) for preparing medicine for treating neoplastic diseases - Google Patents
Application of 2-methoxy-6-acetyl-7-methyljuglone (MAM) for preparing medicine for treating neoplastic diseases Download PDFInfo
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Abstract
The invention discloses application of 2-methoxy-6-acetyl-7-methyljuglone (MAM) for preparing a medicine for treating neoplastic diseases. According to the invention, found from research on the anti-neoplastic activity of MAM, MAM has obvious inhibitory activity on proliferation of a plurality of measured neoplastic cells in vitro; the better cytotoxic effect is shown; the IC50 (50% inhibiting concentration) of MAM is less than or equal to 10 mu M; furthermore, MAM has good dose-effect relationship; simultaneously, when MAM is acted on a normal hepatic cell strain LO2, the cytotoxicity of MAM is lower than the toxic reaction of MAM on a hepatic neoplastic cell HepG2; and furthermore, the research also finds that MAM is capable of obviously reducing mitochondrial membrane potential of neoplastic cells and inducing apoptosis.
Description
Technical field
The present invention relates to natural medicine field, be specifically related to the application of (2-Methoxy-6-Acetyl-7-Methyljuglone also claims 2-Methoxystypandrone, hereinafter to be referred as MAM) of 2-methoxyl group-6-acetyl group-7-methyl juglone.
Background technology
Rhizoma Polygoni Cuspidati is dry rhizome and the root of polygonaceae plant Rhizoma Polygoni Cuspidati (Polygonum cuspidatum Sieb.et Zucc.).Rhizoma Polygoni Cuspidati is traditional Chinese medicine, has the effects such as heat-clearing and toxic substances removing, promoting the function of the gallbladder to alleviate jaundice, expelling wind and removing dampness, dissipating blood stasis analgesic therapy, relieving cough and resolving phlegm.Find the number of chemical composition in the Rhizoma Polygoni Cuspidati, comprised diphenylethylene, Anthraquinones, flavonoid, tannin, polysaccharide etc.Resveratrol (Huang et al., Anticancer Agents Med Chem.2011,11 wherein, 479-490.), emodin (Ma et al., Food Chem Toxicol.2012,50,1271-1278.) etc. be proved and have obvious anti-tumor activity.MAM separates a naphthoquinone compound that obtains from Rhizoma Polygoni Cuspidati, its molecular formula is C
14H
12O
5, molecular weight is 260.2421, is orange acicular crystal, and CAS number is 85122-21-0, and its chemical structural formula is as follows:
Considerably less to the bioactive research of MAM at present.The report MAM such as Singh external have suppress the HRV HRV 3CP active (Singh et al., Bioorg Med Chem Lett.2001,11,3143-3146.); Chiou etc. are reported in nuclear factor KB receptor activation factor aglucon (receptor activator of nuclear factor kappaB (N F-kappaB) ligand, in the osteoclast model of the RAW264.7 cell differentiation of RANKL) inducing, MAM can suppress osteoclast formation, demonstrate effect (the Chiou et al. that certain anti-osteoclast generates, Br J Pharmacol.2010,161,321-335.); Li etc. are reported in external PC12 cell, and the oxidative damage that MAM antagonism tert-butyl group hydrogen peroxide is induced (Li et al., Planta Med.2011,77,354-361.).At present, still there is not the report about the antitumor action of MAM at home and abroad.
Summary of the invention
The object of the present invention is to provide a kind of new medical applications of 2-methoxyl group-6-acetyl group-7-methyl juglone (MAM).
Technical scheme of the present invention is: the application of 2-methoxyl group-6-acetyl group-7-methyl juglone in the medicine of preparation treatment tumor disease, and described 2-methoxyl group-6-acetyl group-7-methyl juglone has structure shown in the structure formula I:
Structural formula I
Described tumor disease is breast carcinoma, pulmonary carcinoma, hepatocarcinoma, colorectal cancer, leukemia, glioma, cancer of pancreas or melanoma.
Described 2-methoxyl group-6-acetyl group-7-methyl juglone separates from Rhizoma Polygoni Cuspidati and obtains.
Described medicine comprises 2-methoxyl group-6-acetyl group-7-methyl juglone and pharmaceutically acceptable carrier.
The present invention finds in the research to the anti-tumor activity of MAM, and it is active that MAM has a significant inhibition in external propagation to the kinds of tumor cells measured, demonstrates preferably cytotoxic effect, its IC
50All be less than or equal to 10 μ M, and have good dose-effect relationship, simultaneously it is acted on normal liver cell strain LO2, its cytotoxicity is lower than it to the toxic reaction of tumor cell of liver HepG2, in addition, find that also MAM can obviously reduce tumor cell mitochondrial membrane potential, cell death inducing.
Description of drawings
Fig. 1, Fig. 2 are respectively the HPLC collection of illustrative plates of given the test agent MAM of the present invention;
Fig. 3 is the amount effect curve of the different cell strain 24h of MAM effect;
Fig. 4 is that MAM is on the form impact of tumor cell MCF-7;
Fig. 5 is that MAM is on the impact of tumor cell MCF-7 detection line mitochondrial membrane potential;
Fig. 6 is MAM inducing tumor cell MCF-7 apoptosis.
The specific embodiment
The present invention provides the application of 2-methoxyl group-6-acetyl group-7-methyl juglone in the medicine of preparation treatment tumor disease, and described 2-methoxyl group-6-acetyl group-7-methyl juglone has structure shown in the structure formula I:
Structural formula I
Above-mentioned 2-methoxyl group-6-acetyl group-7-methyl juglone separates from Rhizoma Polygoni Cuspidati and obtains.Tumor disease wherein is breast carcinoma, pulmonary carcinoma, hepatocarcinoma, colorectal cancer, leukemia, glioma, cancer of pancreas or melanoma.Medicine comprises 2-methoxyl group-6-acetyl group-7-methyl juglone and pharmaceutically acceptable carrier, and pharmaceutical technology routinely makes.
Be further elaborated below in conjunction with the pharmacologically active of specific embodiment to MAM, but the present invention is not limited to this specific examples.
Related reagent used in the present invention, test kit, equipment are commercially available or commonly used, have universality.
Test material
Given the test agent: MAM is that this laboratory obtains by separating in the Polygonum cuspidatum Sieb. et Zucc.Separation method routinely technique carries out: get 5 kilograms of Rhizoma Polygoni Cuspidati, pulverize, with 95% alcohol extraction three times, merge extractive liquid,, be recycled into usefulness petroleum ether and ethyl acetate fractional extraction behind the extractum water suspendible.Ethyl acetate extraction part is through silica gel column chromatography, and chloroform-methanol (100:0 to 95:5) eluting is collected and contained 2-methoxyl group-6-acetyl group-7-methyl juglone component, and recrystallization gets 2-methoxyl group-6-acetyl group-7-methyl juglone sterling.The mass spectrum of sample, hydrogen spectrum and carbon spectrum data and reported in literature data consistent are so determine that structure is 2-methoxyl group-6-acetyl group-7-methyl juglone.Purity by HPLC at Agilent SB C18(4.6x 250mm, 5um) post detects with two kinds of solvent systems, as shown in Figure 1 and Figure 2, purity is greater than 98%.
MAM dimethyl sulfoxide (DMSO, Dimethyl sulfoxide, Sigma, D2650, Hybri-Max
TM, sterile-filtered, BioReagent, suitable for hybridoma, 〉=99.7%) and be mixed with 10mMolL
-1Storage liquid, packing saves backup in-20 ℃.Facing the time spent is diluted to desired concn with culture medium, and the final concentration of DMSO is controlled at below 0.1%, experiment showed, this concentration DMSO on cell without impact.Negative control group adds 100 μ l coordinative solvents, and blank does not add cell.All establish 6 parallel holes for every group.
Cell strain: breast cancer cell MDA-MB-231, lung carcinoma cell 95-D, A549, pancreatic cancer cell MIA PaCa-2, colorectal cancer cells HCT116, melanoma cell SK-MEL-28, adrenal gland's nerve is had a liking for chromium oncocyte PC12, neuroblastoma cell SH-SY5Y, astroglia neuroblastoma cell U87, breast cancer cell MCF-7, MDA-MB-468, lactiferous ducts cancerous cell T47-D, lung carcinoma cell H1299, hepatoma carcinoma cell HepG2, leukaemia K562, B16, normal liver cell strain LO2.Except normal liver cell strain LO2, all the other cells all are purchased from ATCC(American Type Culture Collection, ATCC, the U.S.).MDA-MB-231, MDA-MB-468, SH-SY5Y cell culture are in the DMEM culture fluid that contains 10% hyclone; 95-D, A549, HCT116, MCF-7, T47-D, H1299, HepG2, K562, B16 cell culture are in the RPMI RPMI-1640 that contains 10% hyclone; MIA PaCa-2 cell culture is in the DMEM culture fluid that contains 10% hyclone, 2.5% deactivation horse serum; SK-MEL-28, U87 cell culture are in the EMEM culture fluid that contains 10% hyclone; The PC12 cell culture is in the F-12K culture fluid that contains 15% deactivation horse serum, 2.5% hyclone.All contain 100kU/L penicillin, 100mg/L streptomycin in the culture fluid.Cell is put in 37 ℃, the saturated humidity incubator of 5%CO2 and is cultivated, and changes every other day culture fluid.
Reagent:
The F-12K culture medium (Ham's F-12K,
21127-022)
The DMEM culture medium (Dulbecco's Modified Eagle Medium,
12800-017)
RPMI 1640 culture medium (RPMI 1640 Medium,
23400-021)
EMEM(Eagle's?Minimum?Essential?Medium，ATCC，30-2003)
The FBS(hyclone, Fetal Bovine Serum,
26140-079)
HS (the deactivation horse serum, Horse Serum, Heat-Inactivated,
26050-088)
MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt, Sigma, US19265)
JC-1(5，5’，6，6'-tetrachloro-1，1′，3，3'-tetraethyl-benzimidazolylcarbocyanine?iodide，Molecular
T-31681)
Hoechst?33342(Molecular
H1399)
Instrument:
Microplate reader (Wallac VICTOR3
TMV; Perkin Elmer)
Fluorescence microscope (Carl Zeiss; Axiovert 200)
Camera (Carl Zeiss; AxioCam HRc)
Embodiment 1 (extracorporeal anti-tumor function of MAM)
The take the logarithm tumor cell of trophophase is inoculated in ((5-10) * 10 on 96 orifice plates according to the size of cell
3Individual/hole), behind the 24h that grows, abandon supernatant.Then by following concentration administration, if not dosing group (blank group) and dosing group (concentration 0.1-10 μ M), establish 6 multiple holes for every group, continue to cultivate 24h, abandon supernatant, add the MTT serum-free medium that 100 μ l contain 0.5mg/ml and hatch 4h, abandon supernatant, add 100 μ l dimethyl sulfoxide (DMSO), be positioned over the 10min that vibrates on the miniature concussion instrument, the OD value is detected at microplate reader 570nm place.Be that LO2 does toxicity assessment with the normal liver cell.Each experiment repeats 3 times.The results are shown in Table 1 and Fig. 3.
Fig. 3 shows the IC that the different cell strain 24h of MAM effect MTT detects
50As a result, table 1 is that MAM is to the IC of different tumor cell lines
50(pM) numerical value.
Table 1.
MAM has a liking for the 24h IC of chromium oncocyte PC12, neuroblastoma cell SY5Y, astroglia neuroblastoma cell U87 to breast cancer cell MDA-MB-231, lung carcinoma cell 95-D, A549, pancreatic cancer cell MIA PaCa-2, colorectal cancer cells HCT116, melanoma cell SK-MEL-28, adrenal gland's nerve as can be seen from Table 1
50Between 5-10 μ M; MAM acts on the 24h IC of breast cancer cell MCF-7, MDA-MB-468, lactiferous ducts cancerous cell T47-D, lung carcinoma cell H1299, hepatoma carcinoma cell HepG2, leukaemia K562, B16
50All less than 5 μ M, illustrate that MAM is active better to the inhibition of MCF-7, MDA-MB-468, T47-D, H1299, HepG2, K562 and B16 cell.
Fig. 3 is under different compound concentrations (0.16,0.32,0.625,1.25,2.5,5,10 μ M) to breast cancer cell MCF-7, MDA-MB-468, lactiferous ducts cancerous cell T47-D, lung carcinoma cell H1299, hepatoma carcinoma cell HepG2, leukaemia K562, B16 and the LO2 effect growth inhibited situation after 24 hours, results suggest, in this concentration range, concentration increase along with chemical compound, compare with the blank group, breast cancer cell MCF-7, MDA-MB-468, lactiferous ducts cancerous cell T47-D, lung carcinoma cell H1299, hepatoma carcinoma cell HepG2, leukaemia K562, the proliferation activity of B16 descends respectively, point out this chemical compound to be concentration dependent inhibition tumor cell propagation, and do not suppress the propagation that normal stem cell is the LO2 cell.
Embodiment 2(morphological observation)
Cultivate the MCF-7 cell in 96 orifice plates, be divided into blank group and MAM processed group.The MAM processed group adds the MAM (1 μ M, 2 μ M, 3 μ M, 5 μ M, 8 μ M) of variable concentrations, and the blank group adds the solvent (DMSO) of same concentration, continues to cultivate after 24 hours, examines under a microscope cellular morphology and changes.
As shown in Figure 4, find that blank group cellular morphology is normal, after the MAM of variable concentrations processed, cellular morphology became circle, smaller volume, shrinkage; High concentration MAM effect is lower, cell debris occurs, and cell is floating, and prompting is dead.Concentration dependent is obvious.
Embodiment 3(JC-1 staining examine mitochondrial membrane potential changes)
Cultivate the MCF-7 cell in 24 orifice plates, be divided into blank group and MAM processed group.The MAM processed group adds the MAM (1 μ M, 2 μ M, 5 μ M) of variable concentrations, and the blank group adds the solvent (DMSO) of same concentration.Continue to cultivate after 4 hours, JC-1 dyestuff lucifuge dyeing (final concentration 2 μ g/ml) 15 minutes is taken pictures with fluorescence microscope.
As shown in Figure 5, find that blank group cell has red fluorescence and green fluorescence, and through after the MAM processing, increase along with drug level, green fluorescence increases gradually, red fluorescence reduces gradually, has caused the change of mitochondrial membrane potential in anoxic after prompting MAM processes, and has caused mitochondrial membrane potential decline.
Embodiment 4(Hoechst 33342 staining examine apoptosis)
Cultivate the MCF-7 cell in 24 orifice plates, be divided into blank group and MAM processed group.The MAM processed group adds MAM (the 1 μ M of variable concentrations, 2 μ M, 5 μ M), the blank group adds the solvent DMSO of same concentration, continue to cultivate after 24 hours, through the fixing 20min of 4% paraformaldehyde, PBS washs 2-3 time, Hoechst 33342(final concentration 1 μ g/ml)) lucifuge dyeing is 15 minutes, takes pictures with fluorescence microscope.
As shown in Figure 6, find that blank group cellular morphology is normal, complete through nucleus after the stain dyeing, without obvious dna fragmentation; After variable concentrations MAM processing, karyopyknosis, dna fragmentationization is obvious, and prompting MAM can induce the MCF-7 apoptosis.
Claims (4)
1.2-the application of methoxyl group-6-acetyl group-7-methyl juglone in the medicine of preparation treatment tumor disease, described 2-methoxyl group-6-acetyl group-7-methyl juglone has structure shown in the structure formula I:
Structural formula I
2. application according to claim 1 is characterized in that: described tumor disease is breast carcinoma, pulmonary carcinoma, hepatocarcinoma, colorectal cancer, leukemia, glioma, cancer of pancreas or melanoma.
3. application according to claim 1 is characterized in that: described 2-methoxyl group-6-acetyl group-7-methyl juglone separates from Rhizoma Polygoni Cuspidati and obtains.
4. application according to claim 1 is characterized in that: described medicine comprises 2-methoxyl group-6-acetyl group-7-methyl juglone and pharmaceutically acceptable carrier.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106109450A (en) * | 2016-06-13 | 2016-11-16 | 中山大学 | 2 methoxyl group 6 acetyl group 7 methyljuglone are as indoleamine 2, the purposes of 3 dioxygenase 1 inhibitor |
| KR20180021642A (en) * | 2016-08-22 | 2018-03-05 | 경상대학교산학협력단 | Anti-Helicobacter pylori composition comprising 2-alkoxy-6-acetyl-7-methyljuglone as effective component |
| CN112972442A (en) * | 2021-02-25 | 2021-06-18 | 澳门大学 | New application of 2-methoxy-6-acetyl-7-methyl walnut ketone |
| CN113398107A (en) * | 2021-08-04 | 2021-09-17 | 武汉迦宁科技有限责任公司 | Novel application of 2-benzamido-1, 4-naphthoquinone |
| KR20210136202A (en) * | 2020-05-06 | 2021-11-17 | 전남대학교산학협력단 | Composition for preventing, improving and treating leukemia comprising compound derived from Rumex japonicus houtt roots extract |
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2012
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Non-Patent Citations (1)
| Title |
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| LIU, JIAWEI等: "Small-molecule STAT3 signaling pathway modulators from Polygonum cuspidatum", 《PLANTA MEDICA 》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106109450A (en) * | 2016-06-13 | 2016-11-16 | 中山大学 | 2 methoxyl group 6 acetyl group 7 methyljuglone are as indoleamine 2, the purposes of 3 dioxygenase 1 inhibitor |
| KR20180021642A (en) * | 2016-08-22 | 2018-03-05 | 경상대학교산학협력단 | Anti-Helicobacter pylori composition comprising 2-alkoxy-6-acetyl-7-methyljuglone as effective component |
| KR101961641B1 (en) | 2016-08-22 | 2019-03-25 | 경상대학교산학협력단 | Anti-Helicobacter pylori composition comprising 2-alkoxy-6-acetyl-7-methyljuglone as effective component |
| KR20210136202A (en) * | 2020-05-06 | 2021-11-17 | 전남대학교산학협력단 | Composition for preventing, improving and treating leukemia comprising compound derived from Rumex japonicus houtt roots extract |
| KR102350036B1 (en) * | 2020-05-06 | 2022-01-12 | 전남대학교산학협력단 | Composition for preventing, improving and treating leukemia comprising compound derived from Rumex japonicus houtt roots extract |
| CN112972442A (en) * | 2021-02-25 | 2021-06-18 | 澳门大学 | New application of 2-methoxy-6-acetyl-7-methyl walnut ketone |
| CN113398107A (en) * | 2021-08-04 | 2021-09-17 | 武汉迦宁科技有限责任公司 | Novel application of 2-benzamido-1, 4-naphthoquinone |
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