CN109776478B - iso-Penicillium xanthone A from penicillium oxalicum and application in cervical cancer - Google Patents
iso-Penicillium xanthone A from penicillium oxalicum and application in cervical cancer Download PDFInfo
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- CN109776478B CN109776478B CN201811475759.XA CN201811475759A CN109776478B CN 109776478 B CN109776478 B CN 109776478B CN 201811475759 A CN201811475759 A CN 201811475759A CN 109776478 B CN109776478 B CN 109776478B
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Abstract
The invention relates to iso-Penicillium xanthone A from Penicillium oxalicum and application thereof in cervical cancer. The compound is characterized by comprising the following structural characteristics:
the enantiomers with the known substance Penicillium anthrone A (Penicillium xanthone A). Experiments prove that the anthraquinone compound has better inhibitory activity on cervical cancer cells. Can be used for preparing cervical cancer cell proliferation inhibiting medicine or anti-cervical cancer medicine for anti-tumor research.
Description
Technical Field
The invention relates to iso-Penicillium xanthone A from Penicillium oxalicum and application thereof in cervical cancer, belonging to the field of biological medicine.
Background
With the development of science and technology, the medical level of our medicine has been improved, but the incidence of cancer is on the rise, which is the second leading cause of death worldwide. Cervical cancer is one of the most common cancers in women, and is described by the FDA in the united states as a "silent cancer" that is the leading cancer in female reproductive organs, but also the common cancer in various malignancies in women. 13.5 million new cases in China account for 1/3 of the total disease rate of the world, about 8 million cases die of cervical cancer every year, and the age of the disease is younger in recent years. Cervical cancer is not sensitive to most of the anticancer drugs on the market at present, and the effective rate of chemotherapy is not more than 15%, so that the discovery of novel chemotherapeutic drugs is urgent.
Penicillium anthrone is an anthraquinone organic compound produced by secondary metabolism of fungi, and is first obtained from Penicillium anthrone A in 2002Penicillium thomiiTo date, over a decade has passed. The compounds are reported to be 2 compounds so far, namely penicillanic anthrone A, B and are all connected by 2-4'. The compound has obvious inhibition on tumor cells, and is an ideal raw material for developing antitumor drugs or tumor cell proliferation inhibitors. But studies on the antitumor activity of their enantiomers are still blank.
The present inventors have studied and found that Penicillium oxalicum (Penicillium oxalicum) IBPT-6, (deposited at the chinese type culture collection on 25.12.2013, address: wuhan, wuhan university, the accession number is: CCTCC NO: m2013714) had a good cell proliferation inhibitory activity, and the active ingredients thereof were investigated. Researches show that the penicillanic anthrone compound has anti-human cervical cancer activity, and no report on the proliferation inhibition activity of the compound on human cervical cancer cells exists at present, so that no medicine related to the compound exists in the market.
Disclosure of Invention
The invention aims to provide iso-Penicillium xanthone A derived from penicillium oxalicum and application thereof in cervical cancer.
In order to realize the purpose, the following technical scheme is adopted:
the iso-Penicillium xanthone A derived from Penicillium oxalicum has the effects of inhibiting the proliferation of cervical cancer cells and resisting human cervical cancer activity. The structural formula is as follows:
the preparation method of the compound is to culture the penicillium oxalicum ((C))Penicillium oxalicum) IBPT-6, obtaining fermented mycelium, and separating and purifying the compound from the mycelium. The method comprises the following specific steps:
1 fermentative production
A conventional method for culturing microorganisms comprises collecting Penicillium oxalicum (B) ((R))Penicillium oxalicum) IBPT-6 is inoculated on a flat solid culture medium to be cultured in an incubator at 28 ℃ for 2-3 d, then is inoculated in a fungus culture medium, is statically cultured at 28 ℃ for 30 d, and is filtered by a plurality of layers of gauze to obtain mycelium and fermentation liquor; the fungus culture medium comprises the following components: 10.0 g of glucose, 20.0 g of maltose, 20.0 g of mannitol, 10.0 g of monosodium glutamate, 3.0 g of yeast extract, 15.0 g of NaCl, KH 2 PO 4 0.5 g、MgSO 4 ·7H 2 O0.3 g and ultrapure water 1L.
2. Obtaining extract
The mycelium and the fermentation broth were separated with gauze. Continuously performing ultrasonic wall breaking on the mycelia for 3 times by using an acetone solution (containing 20-30% of water), and filtering to remove residues to obtain a crude extract of the mycelia containing acetone and water. Concentrating under reduced pressure to remove acetone to obtain water solution of crude extract, extracting with ethyl acetate for 3 times at a volume ratio of the water solution to ethyl acetate of 1:2 to obtain ethyl acetate crude extractive solution, concentrating, and evaporating to obtain mycelium extract 36.5 g.
3. Separation and purification of Compound
The mycelium extract is stirred by 100-200 meshes of silica gel, and the mixture is prepared by mixing petroleum ether: dichloromethane: methanol is used as gradient eluent, and decompression silica gel chromatographic column chromatography is carried out. And carrying out simple thin-layer chromatography, merging and separating to obtain the components A-E. Component C (12.7 g) was purified in dichloromethane: methanol =1:2 as gradient eluent, and gel column chromatography is carried out(Sephadex LH-20), and four sub-components C are obtained by combination after thin-layer chromatography analysis 1 -C 4 . Subfraction C 3 (4.9 g) by semi-preparative liquid chromatography (type 1010 ODS-A, 10X 250 mm,5 μm): the separation flow rate was 5 mL/min and the mobile phase was 55% acetonitrile with 0.1% TFA to give the indicated compound (510 mg, t R 22.8 min)。
Said penicillium oxalicum (A), (B)Penicillium oxalicum) IBPT-6, which has been deposited at the China center for type culture Collection on 25.12.2013, address: wuhan, wuhan university, the accession number is: CCTCC NO: m2013714.
The invention also comprises the application of the compound in preparing a medicament for inhibiting the proliferation of human cervical cancer cells and the application of the compound in preparing a medicament for resisting human cervical cancer.
The invention has the following remarkable advantages:
researches show that the penicillanic anthrone compound has obvious activity of inhibiting the proliferation of human cervical cancer cells, and no report of the activity of the compound on the proliferation of the human cervical cancer cells exists at present, so that no medicine related to the activity is found in the market.
Drawings
FIG. 1 major COSY, HMBC and NOE signals of Iso-Penicillixanthone A.
Detailed Description
The chemical structures of the compounds referred to in the examples below:
EXAMPLE 1 fermentative production and isolation purification of the Compound
1 fermentation production
Fermentation culture of producing bacteria: taking Penicillium oxalicum (B) according to a conventional method for culturing microorganismsPenicillium oxalicum) IBPT-6 (deposited at the chinese type culture collection on 25.12.2013, address: wuhan, wuhan university, the accession number is: CCTCC NO: m2013714) in proper amount, inoculating on plate solid culture medium at 28 deg.C2-3 d is cultured in the incubator.
Culturing Penicillium oxalicum 2-3 d in a flat plate (Penicillium oxalicum) An appropriate amount of IBPT-6 was inoculated into a culture medium containing 400 mL [ culture medium composition (g/l): 10.0 percent of glucose, 20.0 percent of maltose, 20.0 percent of mannitol, 10.0 percent of monosodium glutamate, 3.0 percent of yeast extract, 15.0 percent of NaCl and KH 2 PO 4 0.5、MgSO 4 ·7H 2 O0.3, adding ultrapure water to constant volume of 1L, and performing static culture at 28 ℃ for 30 d to obtain mycelium and fermentation liquor.
2. Obtaining extract
The mycelium and the fermentation broth were separated with gauze. Continuously performing ultrasonic wall breaking on the mycelia for 3 times by using an acetone solution (containing 20-30% of water), and filtering to remove residues to obtain a crude extract of the mycelia containing acetone and water. Concentrating under reduced pressure to remove acetone to obtain water solution of crude extract, extracting with ethyl acetate at a volume ratio of the water solution to ethyl acetate of 1:2 for 3 times to obtain ethyl acetate crude extractive solution, and concentrating under reduced pressure to near dry to obtain mycelium extract 36.5 g.
3. Separation and purification of Compound
And (3) mixing the mycelium extract with 100-200 meshes of silica gel, mixing the mixture with petroleum ether: dichloromethane: methanol is used as gradient eluent, and decompression silica gel chromatographic column chromatography is carried out. By simple thin layer chromatography, combining, and separating into components A-E. Component C (12.7 g) was purified as dichloromethane: methanol =1:2 as gradient eluent, performing gel column chromatography (Sephadex LH-20), performing thin layer chromatography, and mixing to obtain four subfractions C 1 -C 4 . Subcomponent C 3 (4.9 g) by semi-preparative liquid chromatography (type 1010 ODS-A, 10X 250 mm,5 μm): the separation flow rate was 5 mL/min and the mobile phase was 55% acetonitrile with 0.1% TFA to give the indicated compound (510 mg, t R 22.8 min)。
A compound: is yellow powder at normal temperature, [ alpha ]] 20 D + 105.6° (c 1, pyridine); [α] 20 D + 3.1° (c 1, Me 2 CO), high-resolution electrospray ionization mass spectrum HRESI-MS at m/z:661.1550 shows the molecular ion peak [ M + Na] + （calcd for C 32 H 30 NaO 14 661.1533); molecular weight at 638, estimated from spectral informationSub-formula is C 32 H 30 O 14 。 1 H and 13 the C-NMR data are shown in Table 1, and the main COSY, HMBC and NOE signals are shown in FIG. 1.
Of the compounds of Table 1 1 H and 13 C-NMR data (500 MHz) 1 H and 126 MHz 13 C, in DMSO-d 6 ）
Example 2 in vitro testing of antitumor Activity
1. Experimental sample and experimental method
Preparing a solution of a sample to be detected: the test sample was the purified compound isolated and purified in example 1. Precisely weighing a proper amount of sample, preparing a mother solution of 100 mM by DMSO, filtering and sterilizing by a filter membrane, and storing at-20 ℃ for later use. The proportion of DMSO is controlled below 1 per mill.
Taking out frozen cell strain from liquid nitrogen, rapidly melting in 37 deg.C water bath, centrifuging at 1200 rpm for 5 min, discarding frozen solution, adding 1 mL culture medium, blowing, sucking into culture bottle, adding 5 mL fresh DMEM or RPMI 1640 culture medium, gently shaking to uniformly suspend cells in the culture medium, placing at 37 deg.C and 5% CO 2 Cultured in an incubator. When the cell density reaches about 90%, passage is carried out, old culture medium is removed, PBS is washed for 2 times, 300 mu L of pancreatin is added (the pancreatin can cover the bottom of the bottle), the cell is digested in an incubator at 37 ℃ until the cell becomes round and is about to fall off, the culture medium containing 10% fetal calf serum is added to stop the digestion, the cell is divided into 2-3 new culture bottles after being blown and evenly mixed, and the culture medium is supplemented to ensure that the cell continues to grow in the incubator.
Cell proliferation inhibitory Activity test method (WST-1 method)
The activity of the anti-tumor cells is evaluated by adopting a WST-1 kit detection method, and the cell concentration is adjusted to be 3 multiplied by 10 after the tumor cells in the logarithmic growth phase are digested 4 a/mL single cell suspension, 100 μ L of the cell suspension was mixed and inoculated into a 96-well plate, 5 replicate wells per concentration. Then placing at 37 ℃ and 5% CO 2 In the incubatorThe culture was carried out overnight. The supernatant was removed and the drug was diluted with medium to different concentrations and added to the corresponding 96-well plates, and the control was added to medium containing the same amount of DMSO. After culturing 72 h, 10. Mu.L of WST-1 solution was added to each well, and after incubating at 37 ℃ for 2 h, the mixture was gently shaken and mixed, and the absorbance at 450 nm was measured using a microplate reader. The mean OD value for 5 wells was calculated and calculated according to the formula: cell proliferation inhibition rate = (OD control group-OD blank group)/(OD experimental group-OD blank group) × 100% the proliferation inhibition rate of each concentration of drug on tumor cells was calculated, and half inhibition rate IC was calculated using Graphpad Prism 5.0 software 50 。
2. Results of the experiment
The results of the cell proliferation inhibitory activity test are shown in Table 2.
TABLE 2 inhibitory Activity of Compounds on human cervical cancer cell proliferation
3. Conclusion
The compound has good anti-tumor activity on human cervical cancer cells. Can be used for preparing cervical cancer cell proliferation inhibition drugs or antitumor drugs for the research of cervical cancer.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (2)
1. The application of a compound in preparing a medicine for inhibiting the proliferation of cervical cancer cells is
，
The compound is prepared by the following method: fermentation of penicillium oxalicumPenicillium oxalicum IBPT-6, obtaining a fermentation product, and then separating and purifying the fermentation product to obtain the compound; wherein said Penicillium oxalicum isPenicillium oxalicum IBPT-6, which has been deposited at the China center for type culture Collection on 25.12.2013, address:wuhan, wuhan university, the accession number is: CCTCC NO: m2013714.
2. The application of a compound in preparing an anti-cervical cancer medicament is
，
The compound is prepared by the following method: fermentation of penicillium oxalicumPenicillium oxalicum IBPT-6, obtaining a fermentation product, and then separating and purifying the fermentation product to obtain the compound; wherein said Penicillium oxalicum isPenicillium oxalicum IBPT-6, which has been deposited at the China center for type culture Collection on 25.12.2013, address: wuhan, wuhan university, the accession number is: CCTCC NO: m2013714.
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| CN103087031A (en) * | 2011-10-28 | 2013-05-08 | 沈阳药科大学 | Application of bistetrahydrobenzopyrone dimer compounds in anti-cancer drugs |
| CN105188741A (en) * | 2013-04-03 | 2015-12-23 | 阿勒丁医疗公司 | Novel nanoparticle compositions |
| CN107298671A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from the secalonic acid H of penicillium oxalicum and prepare the application of anti-human colon cancer drug |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2002065836A2 (en) * | 2001-02-20 | 2002-08-29 | Paul Stamets | Delivery systems for mycotechnologies, mycofiltration and mycoremediation |
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Patent Citations (4)
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|---|---|---|---|---|
| US4424373A (en) * | 1979-01-30 | 1984-01-03 | Asahi Kasei Kogyo Kabushiki Kaisha | Secalonic acids |
| CN103087031A (en) * | 2011-10-28 | 2013-05-08 | 沈阳药科大学 | Application of bistetrahydrobenzopyrone dimer compounds in anti-cancer drugs |
| CN105188741A (en) * | 2013-04-03 | 2015-12-23 | 阿勒丁医疗公司 | Novel nanoparticle compositions |
| CN107298671A (en) * | 2017-06-17 | 2017-10-27 | 福州大学 | Come from the secalonic acid H of penicillium oxalicum and prepare the application of anti-human colon cancer drug |
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| Title |
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| Structure-based discovery of cytotoxic dimeric tetrahydroxanthones;Guangwei Wu 等;《European Journal of Medicinal Chemistry》;20180213;第148卷;第268-278页 * |
| Syntheses of Dimeric Tetrahydroxanthones with Varied Linkages:;Tian Qin 等;《Journal of the American Chemical Society》;20151106;第137卷;第15225-15233页 * |
| 青霉属真菌活性代谢产物研究进展;郑婕施等;《工业微生物》;20171222;第47卷(第06期);第50-56页 * |
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