CN103910746A - Marine fungus-derived Berkeleyones compound, and preparation method and application thereof - Google Patents
Marine fungus-derived Berkeleyones compound, and preparation method and application thereof Download PDFInfo
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- CN103910746A CN103910746A CN201410072112.8A CN201410072112A CN103910746A CN 103910746 A CN103910746 A CN 103910746A CN 201410072112 A CN201410072112 A CN 201410072112A CN 103910746 A CN103910746 A CN 103910746A
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 48
- 229930187955 berkeleyone Natural products 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 241000233866 Fungi Species 0.000 title abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 16
- 229940079593 drug Drugs 0.000 claims abstract description 11
- 241000985522 Penicillium sclerotiorum Species 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 5
- 201000011510 cancer Diseases 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 229940125904 compound 1 Drugs 0.000 claims description 15
- 229940125782 compound 2 Drugs 0.000 claims description 14
- 238000011218 seed culture Methods 0.000 claims description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 241001558929 Sclerotium <basidiomycota> Species 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 239000012531 culture fluid Substances 0.000 claims description 7
- 238000011275 oncology therapy Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 235000002639 sodium chloride Nutrition 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000013375 chromatographic separation Methods 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 229960001866 silicon dioxide Drugs 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 230000001093 anti-cancer Effects 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 206010009944 Colon cancer Diseases 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 201000007270 liver cancer Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 239000000401 methanolic extract Substances 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 2
- 210000004907 gland Anatomy 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 8
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 2
- 230000004663 cell proliferation Effects 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 230000002700 inhibitory effect on cancer Effects 0.000 abstract 1
- 241000894007 species Species 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 240000002044 Rhizophora apiculata Species 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000012916 structural analysis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 0 CC(C1(OC)OC(C2(C)C(C3)C4C)C=C(C(C)(C)O5)C4=CC5=O)OC4OC5OC5C3(C)C4C12C(*)=O Chemical compound CC(C1(OC)OC(C2(C)C(C3)C4C)C=C(C(C)(C)O5)C4=CC5=O)OC4OC5OC5C3(C)C4C12C(*)=O 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000228150 Penicillium chrysogenum Species 0.000 description 1
- 240000003793 Rhizophora mangle Species 0.000 description 1
- HNVJWWBKFFDQAA-NRKPLWJESA-N [(7r,8r,8ar)-7-hydroxy-7-methyl-6-oxo-3-[(e)-prop-1-enyl]-8,8a-dihydro-1h-isochromen-8-yl] 2,4-dihydroxy-6-methylbenzoate Chemical class O([C@H]1[C@@](C)(O)C(=O)C=C2C=C(OC[C@@H]21)/C=C/C)C(=O)C1=C(C)C=C(O)C=C1O HNVJWWBKFFDQAA-NRKPLWJESA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000013043 cell viability test Methods 0.000 description 1
- 230000006364 cellular survival Effects 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000010304 tumor cell viability Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
-
- 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
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of drug compounds and specifically relates to a marine fungus-derived Berkeleyones compound and a preparation method and application thereof. The structure of the Berkeleyones compound is represented by a formula (I) as described in the specification; the novel compound has an inhibitory effect on cancer cell proliferation and can be used for preparing an anticancer drug. The Berkeleyones compound is separated from the marine fungus Penicillium sclerotiorum SJ0167. There are a variety of species of and a great number of marine microorganisms; the method for extracting the Berkeleyones compound from marine microorganisms is simple and comprises easy and convenient steps; thus, the Berkeleyones compound is widely available and has low cost, and the Berkeleyones compound has high inhibitory activity on cancer cells and wide application prospects.
Description
Technical field
The invention belongs to medical compounds technical field, be specifically related to the Berkeleyones compound in a class thalassiomycetes source and its preparation method and application.
Background technology
Mangrove forest is distinctive evergreen shrubs and dungarunga group on the torrid zone, bay, subtropics, estuary mud bar, has respiratory root or stilit root, is generally distributed in the tideland between high water mark and subtidal line.In China, mangrove forest is mainly distributed in Hainan Island, Guangxi, Guangdong and Fujian.As the second largest monoid in thalassiomycetes, mangrove fungi is due to growth conditions uniqueness, and active metabolite is very abundant, aspect marine resources development, has great significance.Current scientist isolates a lot of novel structures, has the compound of remarkable activity, many clinical experimental stages that entered from the various endogenous fungus metabolites of its inside.
Berkeleyones compound derives from the Penicillium notatum separating from the mine lake of Berkeley at first, this bacterial strain still can be survived under rugged environment, its meta-bolites probably has active substance, therefore researchist has conducted in-depth research and has found the Berkeleyones compound of new texture, and think that this compounds plays an important role (Donald B. Stierle, etal. Org. Lett., 2004 in fungus body, 6,1049-1052).What the compound of this class formation had been reported at present only has 15 (Donald B. Stierle, etal. J. Nat. Prod., 2007,70,1820-1823; Donald B. Stierle, etal. J. Nat. Prod., 2011,74,2273-2277; Motoo lida, etal. Org. Lett., 2008,10,845-848; Yi Zhang, etal. J. Nat. Prod., 2012,75,1888-1895).
Cancer is serious threat human life's common disease and frequently-occurring disease.Research and development are efficient, the new type anticancer medicine of low toxicity, high specificity is the important directions of current antitumor drug research.Marine natural product, as one of important sources of lead compound, has important effect to the research and development of newtype drug.
Summary of the invention
The object of the present invention is to provide the Berkeleyones compound in the thalassiomycetes source that a class is new.Described Berkeleyones compound is from a kind of South Sea mangrove endophytic fungus sclerotium mould
penicillium sclerotiorum.SJ separate in 0167 and to obtain, this compound all shows good antitumour activity to breast cancer cell (MDA-MB-435), liver cancer cell (HepG2), colon cancer cell (HCT-116) and lung adenocarcinoma cell (A549), can be applicable to prepare cancer therapy drug.
Another object of the present invention is to provide the preparation method of above-mentioned Berkeleyones compound.
A further object of the invention is to provide the application of above-mentioned Berkeleyones compound.
Above-mentioned purpose of the present invention is achieved by following technical solution:
One class Berkeleyones compound, its structural formula as shown in the formula (I):
。
The fungi sclerotium mould that the present invention is used
penicillium sclerotiorum.SJthe 0167th, from isolated a kind of endogenetic fungus in the mangrove forest of the South Sea, Classification And Nomenclature is sclerotium mould
penicillium sclerotiorum, this bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and preservation date is on December 23rd, 2013, and preserving number is CGMCC NO:8628; Depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The preparation method of above-mentioned Berkeleyones compound, comprises the steps:
S1. fungi
penicillium sclerotiorum.SJ 0167 bacterial strain access seed culture medium, shaking table is cultivated, and obtains seed culture fluid;
S2. seed culture fluid is accessed in fermention medium, leave standstill and cultivate to obtain fermented product;
S3. fermented product is soaked and extracted with methyl alcohol, after methanol extract liquid is concentrated, through ethyl acetate extraction, concentrated, obtains medicinal extract, then through chromatographic separation, obtain Berkeleyones compound 1 and compound 2 shown in formula (I).
Described in step S1, seed culture medium adopts the GYT substratum of this area routine, incubated at room temperature 5 ~ 7 days; The composition of conventional GYT substratum is by weight: glucose 18 ~ 23g, peptone 4 ~ 5g, yeast extract paste 1 ~ 2g, sea salt 55 ~ 65g, water 1.5 ~ 2L.Described in step S1, shaking table cultivation is under room temperature, shaking speed 100 ~ 150rpm, and incubation time is 5 ~ 7 days.
As a kind of preferred version, in above-mentioned preparation method, fermention medium is solid rice medium described in step S2, and its component is: rice 50 ~ 70g, sea salt 1.5 ~ 2g, water 50 ~ 70mL.Described in step S2, leaving standstill the time of cultivating is 1 ~ 2 month, and leaving standstill the temperature of cultivating is room temperature.
As a kind of preferred version, in above-mentioned preparation method, the consumption of methyl alcohol is and fermented product equal-volume described in step S3, and the consumption of ethyl acetate is methanol usage 1/3; Described medicinal extract carries out chromatographic separation with silicagel column, uses respectively the petroleum ether-ethyl acetate gradient elution of 10:0,9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9 and 0:10 when silicagel column carries out chromatographic separation.The petroleum ether-ethyl acetate wash-out part of 7:3,6:4 and 5:5 is merged, through dextrane gel Sephadex LH-20 chromatography, sherwood oil-the methylene chloride-methanol that is 2:1:1 by volume ratio is the further wash-out of eluent, pass through again HPLC, the methanol-water that is 6:4 by volume ratio is that eluent is purified, and obtains compound 1 and compound 2 shown in formula (I).
Described silicagel column is the silicagel column of this area routine, and order number is about 200 ~ 300 orders.
The present invention separates the Berkeleyones compound obtaining and has the effect that anticancer is bred, and therefore can be used for preparing cancer therapy drug.
Described anticancer anti-breast cancer, anti-liver cancer, inhibitor against colon carcinoma cells or the anti-lung gland cancer of comprising.
The present invention has following beneficial effect:
New skeleton Berkeleyones compound of the present invention is to separate and obtain from a kind of thalassiomycetes, and Marine Microbial Kinds is various, and quantity is huge, the method of extracting compound from microorganism is simple, step is easy, makes Berkeleyones compound source abundant, with low cost; Described new skeleton Berkeleyones compound has the effect of anticancer propagation, can be used for preparing cancer therapy drug, has a extensive future.
Brief description of the drawings
Fig. 1 is the proton nmr spectra of Berkeleyones compound 1 of the present invention.
Fig. 2 is the carbon-13 nmr spectra of Berkeleyones compound 1 of the present invention.
Fig. 3 is the nucleus magnetic resonance H of Berkeleyones compound 1 of the present invention, H-cosy two-dimensional spectrum.
Fig. 4 is the nucleus magnetic resonance HSQC two-dimensional spectrum of Berkeleyones compound 1 of the present invention.
Fig. 5 is the nucleus magnetic resonance HMBC two-dimensional spectrum of Berkeleyones compound 1 of the present invention.
Fig. 6 is the nucleus magnetic resonance NOE two-dimensional spectrum of Berkeleyones compound 1 of the present invention.
Fig. 7 is the proton nmr spectra of Berkeleyones compound 2 of the present invention.
Fig. 8 is the carbon-13 nmr spectra of Berkeleyones compound 2 of the present invention.
Fig. 9 is the nucleus magnetic resonance H of Berkeleyones compound 2 of the present invention, H-cosy two-dimensional spectrum.
Figure 10 is the nucleus magnetic resonance HSQC two-dimensional spectrum of Berkeleyones compound 2 of the present invention.
Figure 11 is the nucleus magnetic resonance HMBC two-dimensional spectrum of Berkeleyones compound 2 of the present invention.
Figure 12 is the nucleus magnetic resonance NOE two-dimensional spectrum of Berkeleyones compound 2 of the present invention.
Embodiment
Below in conjunction with Figure of description and specific embodiment, the present invention is further explained, but embodiments of the present invention is not limited in any way.Unless stated otherwise, in embodiment, related reagent, method is the conventional reagent in this area and method.
extraction and the sign of embodiment 1 compound
Compound of the present invention, can be from South Sea mangrove endophytic fungus sclerotium mould
penicillium sclerotiorum.SJ separate in 0167 and obtain, thalassiomycetes sclerotium mould
penicillium sclerotiorum.SJ 0167 is to separate and obtain from the Hai Sang of Shenzhen.This bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, preservation date is on December 23rd, 2013, preserving number is CGMCC NO:8628, and depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The concrete preparation process of compound is as follows:
S1. the acquisition of seed culture fluid:
S11. prepare seed culture medium: glucose 20g, peptone 4g, yeast extract paste 2g, sea salt 60g, tap water 2000mL, average mark is loaded on 8 500mL Erlenmeyer flasks, and 121 DEG C go out 25 minutes.
S12. the cultivation of seed: by thalassiomycetes
penicillium sclerotiorum.SJ 0167 bacterial strain access seed culture medium, at the temperature of 28 DEG C, puts the rotating speed with 120rpm on shaking table, cultivates 72 hours to obtain seed culture fluid.
S2. fermentation culture: the in-built 60 g rice of 1000 mL triangular flasks, 2g sea salt, 60mL water, the seed culture fluid access under Bechtop aseptic technique, 5mL step S1 being obtained after 121 DEG C of (0.1 MPa) high-temperature sterilization 25 min is equipped with in the Erlenmeyer flask of fermention medium, inoculate altogether 90 bottles, leave standstill and cultivate 30 days to obtain fermented product in room temperature.
S3. the extraction of Berkeleyones compound separates: cultured step S2 fermented product, with every bottle of 150 mL methanol extraction three times, is obtained to methanol extract; Methanol extract obtains enriched material through concentrated, and enriched material extracts 3 times by ethyl acetate, and each consumption is every bottle of 50mL, concentrates to obtain crude extract medicinal extract 67 g.This crude extract medicinal extract carries out chromatographic separation with 200 ~ 300 object silicagel columns, is specially the petroleum ether-ethyl acetate gradient elution of using respectively 10:0,9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9 and 0:10.The petroleum ether-ethyl acetate wash-out part of 7:3,6:4 and 5:5 is merged, through dextrane gel Sephadex LH-20 chromatography, sherwood oil-the methylene chloride-methanol that is 2:1:1 by volume ratio is the further wash-out of eluent, pass through again HPLC, the methanol-water that is 6:4 by volume ratio is that eluent is purified, and obtains formula (I) Berkeleyones compound 1(20 mg) and 2(10 mg).
The compound 1 of separation and Extraction is white solid, and the spectrogram that it is carried out to nuclear magnetic resonance spectroscopy detection is as shown in Fig. 1 ~ 6.
The physico-chemical property data of compound 1 structural analysis test are as follows:
ESIMS
?m/z?489.2?[M+H]
+,?487.2?[M-H]
-,HRESIMS?
m/z?488.2048?(calcd?for?C
26H
32O
9?488.2041)。
1H?NMR?(500?MHz,?CDCl
3)?δ?6.50?(s,?1H),?6.11?(s,?1H),?5.85?(s,?1H),?5.53?(d,?
J?=?7.5?Hz,?1H),?4.54?(s,?1H),?3.82?(s,?3H),?3.63?(q,?
J?=?6.1?Hz,?1H),?3.03?(d,?
J?=?7.6?Hz,?1H),?2.79?(m,?1H),?1.79?(d,?
J?=?12.5?Hz,?1H),?1.71?(d,?
J?=?13.2?Hz,?1H),?1.65?(d,?
J?=?12.7?Hz,?1H),?1.63?(s,?3H),?1.48?(s,?6H),?1.35?(d,?
J?=?6.1?Hz,?3H),?1.16?(d,?
J?=?7.6?Hz,?3H),?0.99?(s,?3H)；
13C?NMR?(126?MHz,?CDCl
3)?δ?180.3?(C),?175.3?(C),?163.3?(C),?157.6?(C),?134.7?(C),?129.5?(CH),?120.5?(CH),?102.7?(C),?99.0?(CH),?82.2?(C),?80.7?(CH),?77.6?(CH),?61.0?(C),?52.7?(CH
3),?49.1?(C),?46.6?(C),?45.0?(CH),?43.7?(CH),?43.4?(CH),?34.70?(CH
2),?27.2?(CH
3),?26.6?(CH
3),?26.1?(CH
3),?18.6?(CH
3),?17.4?(CH
3),?13.6?(CH
3)。
The compound 2 of separation and Extraction is white solid, and the spectrogram that it is carried out to nuclear magnetic resonance spectroscopy detection is as shown in Fig. 7 ~ 12.
The physico-chemical property data of compound 2 structural analysis tests are as follows:
ESIMS
?m/z?489.2?[M+H]
+,?487.1?[M-H]
-,HRESIMS?
m/z?488.2048?(calcd?for?C
26H
32O
9?488.2041)。
1H?NMR?(500?MHz,?CDCl
3)?δ?5.91?(d,?
J?=?7.2?Hz,?1H),?5.85?(s,?1H),?4.24?(dd,?
J?=?10.2,?6.2?Hz,?1H),?3.90?(s,?3H),?3.78?(q,?
J?=?6.2?Hz,?1H),?3.36?(dt,?
J?=?19.8,?2.7?Hz,?1H),?3.06?(d,?
J?=?2.4?Hz,?1H),?3.04?(d,?
J?=?9.8?Hz,?1H),?2.99?(d,?
J?=?14.6?Hz,?1H),?2.85?(d,?
J?=?19.1?Hz,?1H),?2.44?(d,?
J?=?6.8?Hz,?1H),?2.40?(d,?
J?=?8.7?Hz,?1H),?1.91?(s,?3H),?1.52?(s,?3H),?1.50?(s,?3H),?1.46?(s,?3H),?1.42?(d,?
J?=?6.2?Hz,?3H),?1.16?(s,?3H)；
13C?NMR?(126?MHz,?CDCl
3)?δ?177.4?(C),?175.0?(C),?170.6?(C),?134.8?(C),?132.5?(C),?128.3?(C),?126.1?(C),?100.7?(C),?98.4?(CH),?84.3?(C),?79.4?(CH),?73.2?(CH),?62.1?(C),?53.3?(CH
3),?50.5?(C),?46.2?(C),?44.8?(CH),?35.0?(CH
2),?34.1?(CH
2),?34.0?(CH
2),?26.8?(CH
3),?26.3?(CH
3),?26.2?(CH
3),?19.1?(CH
3),?14.9?(CH
3),?13.7?(CH
3)。
Molecular formula that can deterministic compound 1 from the structural analysis detected result of nucleus magnetic resonance is C
26h
32o
9, the molecular formula of compound 2 is C
26h
32o
9, structural formula as shown in the formula (I):
。
the antitumour activity test of embodiment 2 compounds
That the antitumour activity test of compound adopts is mtt assay (T. Mosmann. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. Journal of immunological methods.
journal of Immunological Methods 1983,65,55-63.).
(1) material
Four Cuo salt (MTT): with phosphate buffered saline buffer (PBS) the dissolving MTT (3-4,5-dimethythiazol-z-yl) 2 of 0.01mol/L, 5-diphenytetrazolium bromide, SIGMA), ultimate density 5mg/ml, filtration sterilization, after packing, 4 DEG C keep in Dark Place.
The sodium laurylsulfonate of the preparation of MTT lysate: 80g is dissolved in the N-N-dimethyl formamide of 200ml, and heating in water bath hydrotropy, adds 200ml distilled water, mixes adjust pH to 4.7 with 80% acetic acid with 1N hydrochloric acid (1:1).
Cell strain is selected: MDA-MB-435, HepG2, HCT-116, A549 tumor cell line.5% CO at 37 DEG C
2preservation in the air of content.
(2) operation steps
Above four kinds of tumour cells in logarithmic phase are inoculated in respectively to 96 orifice plates, use Dulbecco ' s modified Eagle ' s medium (DMEM) perfect medium by cell dilution to 1 × 10
4individual/ml, the cell that every hole adds 200 μ L to dilute, every group of five Duplicate Samples, separately establish blank well and control wells, and described blank well is the hole of inoculating cell not, and described control wells is the nutrient solution containing medicine not.At 5%CO
2in, under 37 DEG C of room temperatures and saturated humidity, cultivate 24 hours.Remove substratum, (compound method of the cancer therapy drug solution of described different concns is for first making mother liquid medicine with a small amount of DMSO dissolved substance to add the cancer therapy drug solution of different concns, with DMEM perfect medium, mother liquid medicine being diluted to medicine final concentration is again 0, 0.1, 0.5, 1, 5, 10, 20, 30, 40, the solution of the Azaphilone class dimer compound of the present invention of 50 μ M, in drug solution after dilution, the volume percent of DMSO is not higher than 0.1 % of cumulative volume), every hole 200 μ L, cultivate 48 hours, every hole adds the MTT(Sigma of 2mg/ml) 20 μ L, hatch 4 hours.As far as possible nutrient solution in sucking-off hole completely, adds DMSO liquid (150 μ L/ hole), vibrates 10 minutes, and crystallisate is fully dissolved.Under 570nm wavelength, measure each hole OD value by microplate reader; To the mapping of drug level logarithm, obtain IC with absorbance
50value, result represents with mean value ± standard deviation.
Compound of the present invention carries out all showing the restraining effect to cancer cells in 4 kinds of tumor cell viability tests, and test result is as shown in table 1 below.
Table 1
| IC 50 (μg/mL) | Mammary cancer MDA-MB-435 | Liver cancer HepG2 | Colorectal carcinoma HCT-116 | Adenocarcinoma of lung A549 |
| Compound 1 | 34.250±1.59 | 24.565±2.74 | 33.719±3.21 | 37.819±0.39 |
| Compound 2 | 31.316±2.63 | 23.869±1.79 | 29.190±2.37 | 34.0573±1.17 |
Claims (10)
1. a Berkeleyones compound, is characterized in that, its structural formula as shown in the formula (I):
。
2. the preparation method of Berkeleyones compound described in claim 1, is characterized in that, comprises the steps:
S1. by thalassiomycetes sclerotium mould
penicillium sclerotiorum.SJ 0167 bacterial strain access seed culture medium, shaking table is cultivated, and obtains seed culture fluid;
S2. seed culture fluid is accessed in fermention medium, leave standstill and cultivate to obtain fermented product;
S3. fermented product is soaked and extracted with methyl alcohol, after methanol extract liquid is concentrated, through ethyl acetate extraction, concentrated, obtain medicinal extract, medicinal extract, again through chromatographic separation, obtains the compound 1 shown in formula (I) and compound 2;
Described sclerotium mould
penicillium sclerotiorum.SJ 0167 bacterial strain is in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, and preservation date is on December 23rd, 2013, and preserving number is CGMCC NO:8628.
3. the preparation method of Berkeleyones compound according to claim 2, is characterized in that, the component of seed culture medium is described in step S1: glucose 18 ~ 23g, peptone 4 ~ 5g, yeast extract paste 1 ~ 2g, sea salt 55 ~ 65g, water 1.5 ~ 2L.
4. the preparation method of Berkeleyones compound according to claim 2, is characterized in that, to cultivate be under room temperature to shaking table described in step S1, shaking speed 100 ~ 150rpm, and incubation time is 5 ~ 7 days.
5. the preparation method of Berkeleyones compound according to claim 2, is characterized in that, the component of fermention medium is described in step S2: rice 50 ~ 70g, sea salt 1.5 ~ 2g, water 50 ~ 70mL.
6. the preparation method of Berkeleyones compound according to claim 2, is characterized in that, leaving standstill the time of cultivating described in step S2 is 1 ~ February, and leaving standstill the temperature of cultivating is room temperature.
7. the preparation method of Berkeleyones compound according to claim 2, is characterized in that, the consumption of methyl alcohol is and fermented product equal-volume described in step S3; The consumption of ethyl acetate is 1/3 of methanol usage; Described medicinal extract separates with silica gel column chromatography, uses respectively the petroleum ether-ethyl acetate gradient elution of 10:0,9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9 and 0:10 when silicagel column carries out chromatographic separation.
8. the preparation method of Berkeleyones compound according to claim 7, it is characterized in that, the petroleum ether-ethyl acetate wash-out part of 7:3,6:4 and 5:5 is merged, through dextrane gel Sephadex LH-20 chromatography, sherwood oil-the methylene chloride-methanol that is 2:1:1 by volume ratio is the further wash-out of eluent, pass through HPLC, the methanol-water that is 6:4 by volume ratio is that eluent is purified, and obtains the compound 1 shown in formula (I) and compound 2 again.
Described in claim 1 Berkeleyones compound in the application of preparing in cancer therapy drug.
10. Berkeleyones compound, in the application of preparing in cancer therapy drug, is characterized in that according to claim 9, described anticancer anti-breast cancer, anti-liver cancer, inhibitor against colon carcinoma cells or the anti-lung gland cancer of comprising.
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