CN108892658A - Compound lithocarpinol B and preparation method thereof and preparing the application in antifungal drug - Google Patents
Compound lithocarpinol B and preparation method thereof and preparing the application in antifungal drug Download PDFInfo
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- CN108892658A CN108892658A CN201810974840.6A CN201810974840A CN108892658A CN 108892658 A CN108892658 A CN 108892658A CN 201810974840 A CN201810974840 A CN 201810974840A CN 108892658 A CN108892658 A CN 108892658A
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- lithocarpinol
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 38
- 239000003429 antifungal agent Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 241000228197 Aspergillus flavus Species 0.000 claims abstract description 18
- 241000461379 Diaporthe lithocarpus Species 0.000 claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 238000000855 fermentation Methods 0.000 claims description 16
- 230000004151 fermentation Effects 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000000284 extract Substances 0.000 claims description 15
- 239000003208 petroleum Substances 0.000 claims description 15
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 14
- 238000004809 thin layer chromatography Methods 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 241000209094 Oryza Species 0.000 claims description 8
- 235000007164 Oryza sativa Nutrition 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 235000009566 rice Nutrition 0.000 claims description 8
- 235000002639 sodium chloride Nutrition 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 244000061456 Solanum tuberosum Species 0.000 claims description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 6
- 239000007864 aqueous solution 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
- 239000000706 filtrate Substances 0.000 claims description 6
- 230000014759 maintenance of location Effects 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004821 distillation Methods 0.000 claims description 5
- 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 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- UGAPHEBNTGUMBB-UHFFFAOYSA-N acetic acid;ethyl acetate Chemical compound CC(O)=O.CCOC(C)=O UGAPHEBNTGUMBB-UHFFFAOYSA-N 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000002024 ethyl acetate extract Substances 0.000 claims description 3
- 239000002054 inoculum Substances 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
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 229960001866 silicon dioxide Drugs 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000012549 training Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 1
- 239000002609 medium Substances 0.000 claims 1
- 235000015097 nutrients Nutrition 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 241000233866 Fungi Species 0.000 abstract description 5
- 230000000843 anti-fungal effect Effects 0.000 abstract description 4
- 238000009509 drug development Methods 0.000 abstract description 4
- 230000001857 anti-mycotic effect Effects 0.000 abstract description 3
- 239000002543 antimycotic Substances 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 3
- 239000002547 new drug Substances 0.000 abstract description 3
- 229940125904 compound 1 Drugs 0.000 description 23
- 238000001228 spectrum Methods 0.000 description 6
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 3
- 229960000988 nystatin Drugs 0.000 description 3
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 3
- 239000001965 potato dextrose agar Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 238000001026 1H--1H correlation spectroscopy Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 150000005829 chemical entities Chemical class 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001633106 Lithocarpus Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001480007 Phomopsis Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000002115 aflatoxin B1 Substances 0.000 description 1
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 1
- 229930020125 aflatoxin-B1 Natural products 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- KVFIJIWMDBAGDP-UHFFFAOYSA-N ethylpyrazine Chemical compound CCC1=CN=CC=N1 KVFIJIWMDBAGDP-UHFFFAOYSA-N 0.000 description 1
- 235000011868 grain product Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- PQNFLJBBNBOBRQ-UHFFFAOYSA-N indane Chemical class C1=CC=C2CCCC2=C1 PQNFLJBBNBOBRQ-UHFFFAOYSA-N 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000013332 literature search Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
- C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
- C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
- C07D319/20—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring with substituents attached to the hetero ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- 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
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- Organic Chemistry (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses compound lithocarpinol B and preparation method thereof and preparing the application in antifungal drug.The present invention by the fermented and cultured of deep-sea fungi Phomopsis lithocarpus FS508, tunning isolate and purify and Structural Identification, obtain 12 with antifungal activity, 3-dihydro-1H-indene class noval chemical compound, is named as lithocarpinol B.The compound has good inhibitory activity to aspergillus flavus, can be used for preparing antifungal drug.Therefore, the present invention provides candidate compound for antimycotic new drug development, provides scientific basis for development and utilization marine fungi resource.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to compound lithocarpinol B and preparation method thereof
With preparing the application in antifungal drug.
Background technique
Aspergillus flavus, a kind of common saprophytic fungus are more common on the organic matter of mouldy grain, grain product and other mould corruption,
It is 1 class carcinogenic substance that its generated toxin delimited by the Agency for Research on Cancer of the World Health Organization (WHO), is that a kind of toxicity is extremely strong
Extremely toxic substance.Toxin caused by aspergillus flavus is the most common with aflatoxin B1, and toxicity and carcinogenicity are also most strong, to people
And animal's liver group is woven with destruction, and when serious, it is even dead to can lead to liver cancer.Therefore, novel aspergillus flavus resisting medicine is found
Object is of great significance.
Ocean accounts for about the 71% of earth surface product, contains microbial resources abundant, special marine environment (low temperature, height
Pressure, with high salt, high and cold, oligotrophic etc.) and marine species between Ecology Action, assign marine microorganism generate be different from the micro- life in land
The special outcome of object, including antibacterial, anti-phytopathogen, antitumor, antiviral isoreactivity substance.The secondary generation of marine microorganism
Thank to product with unique structure type and rich and varied bioactivity.These new chemical entities can not only disclose new biology
Route of synthesis, and people can also be inspired to carry out bio-mimetic syntheses and bioactivity research to these chemical entities.Therefore, ocean is micro-
Biological secondary metabolite has important scientific research and application value, is the valuable source of drug development.
Deep-sea fungi Phomopsis lithocarpus FS508 be in May, 2016 from the Indian Ocean (16 ° of 50.508'N,
111 ° of 53.335'E, 3606 meters of the depth of water) at it is isolated in deposit on marine-bottom surface.Literature Consult shows up to the present do not have
It is reported about the correlative study of Phomopsis lithocarpus FS508 metabolite antifungal activity.
Summary of the invention
The first purpose of the invention is to provide the noval chemical compound lithocarpinol B to fungi with inhibitory activity.
The compound of the present invention lithocarpinol B, shown in structure such as formula (I)：
A second object of the present invention is to provide the preparation method of compound lithocarpinol B a kind of, the preparation sides
Compound lithocarpinol B described in method is the fermentation from deep-sea fungi Phomopsis lithocarpus FS508
Separation is prepared in culture, specifically includes following steps：
(1) fermentation training is extracted with ethyl acetate in the fermentation culture medium for preparing Phomopsis lithocarpus FS508
Object is supported, acetic acid ethyl acetate extract is obtained into medicinal extract after distillation and concentration；It is dissolved and is soaked with methanol aqueous solution (volume fraction 85%)
Then cream uses petroleum ether extraction, to remove low polar fatty acid material, remaining methanol aqueous solution part distillation and concentration is obtained
Brownish black medicinal extract；
(2) brownish black medicinal extract is crossed into normal phase silicagel column, first uses petroleum ether-ethyl acetate from 10:1,7:1,9:2,3:1,2:
1,1:1,1:2v/v gradient elution, then eluted with ethyl acetate, finally use methylene chloride：Methanol=5:1,1:1v/v gradient is washed
It is de-；Medicinal extract is collected by petroleum ether：Ethyl acetate volume ratio 3:The fraction F6 of 1 elution, component F6 through Sephadex LH-20 column, with
Methylene chloride：Methanol=1:1v/v collects TLC thin-layer chromatography as eluant, eluent with petroleum ether：Ethyl acetate=2:1v/v expansion
Rf=3.0/3.8 component, normal-phase silica gel column chromatography is then crossed, with petroleum ether-ethyl acetate from 6:1,3:1,2:1,1:1,
1:2,0:1v/v gradient elution collects TLC thin-layer chromatography with methylene chloride：Methanol=20:Rf=3.3/3.6 is unfolded to obtain in 1v/v
Component, then the component is separated with full preparation HPLC, the chromatographic condition for preparing HPLC entirely is：YMC ODS-A column, mobile phase are volume
Than 85:15 methanol/water, flow velocity 8mL/min collect eluting fraction at retention time 14.8min, are finally prepared with half
HPLC purifies the component, and the chromatographic condition for partly preparing HPLC is：YMC-pack ODS-A/AQ column, mobile phase are volume ratio 82:18
Acetonitrile/water, flow velocity 3mL/min, retention time 9.4min obtain compound lithocarpinol B.
The fermentation culture medium for preparing Phomopsis lithocarpus FS508 is the Phomopsis that will be activated
Lithocarpus FS508 strain is linked into potato dextrose broth, and 28 DEG C, 120rpm, obtained kind of culture 5d
Seed liquor is linked into rice solid fermentation culture medium, at room temperature stationary culture one by sub- liquid with the inoculum concentration of 10%mL/g
Month, fermentation culture medium is made.
The potato dextrose broth obtains in the following manner：Potato is cleaned into peeling, is cut into size
About 1cm3Fritter, weigh 200g, be put into 1L water and boil 20~30 minutes, filtered with double gauze, take filtrate, in filtrate
Middle addition sea salt 15g, glucose 20g, add water to supply 1L, and sterilizing is made.
The rice solid fermentation culture medium obtains in the following manner：3g sea salt and 160g rice are weighed respectively, are added
200mL water, is stirred, and sterilizing is made.
Third object of the present invention is to provide deep-sea fungi Phomopsis lithocarpus FS508 to prepare chemical combination
Application in object lithocarpinol B.
The present invention is tested by antifungal activity and is found, compound lithocarpinol B has fungi aspergillus flavus bright
Aobvious inhibitory activity, the MIC value to aspergillus flavus are 20 μ g/mL, and positive control medicine nystatin is 15 μ to aspergillus flavus MIC value
g/mL.The result shows that：The compound of the present invention lithocarpinol B can be used to make to the apparent inhibitory activity of aspergillus flavus
Standby antifungal drug.
Fourth object of the present invention is to provide compound lithocarpinol B and is preparing answering in antifungal drug
With the antifungal drug is preferably the drug of aspergillus flavus resisting.
Fifth object of the present invention is to provide a kind of antifungal drug, which contains compound
Lithocarpinol B is as effective component；The antifungal drug is preferably the drug of aspergillus flavus resisting.
Compared with prior art, advantage of the invention is that：
The present invention passes through the fermented and cultured of deep-sea fungi Phomopsis lithocarpus FS508, point of tunning
From purifying and Structural Identification, 12,3-dihydro-1H-indene class noval chemical compound with antifungal activity, life are obtained
Entitled lithocarpinol B.The compound has good inhibitory activity to aspergillus flavus, can be used for preparing antifungal drug.
Therefore, the present invention provides candidate compound for antimycotic new drug development, provides scientific basis for development and utilization marine fungi resource.
Deep-sea fungi Phomopsis lithocarpus FS508 of the invention was preserved in Guangdong on August 15th, 2018
It saves Culture Collection (GDMCC), address：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, deposit number are：
GDMCC NO：60433.
Detailed description of the invention
Fig. 1 is compound 1 (lithocarpinol B)1H-NMR spectrum.
Fig. 2 is compound 1 (lithocarpinol B)13C-NMR spectrum.
Fig. 3 is the hsqc spectrum of compound 1 (lithocarpinol B).
Fig. 4 is compound 1 (lithocarpinol B)1H-1H COSY spectrum.
Fig. 5 is the HMBC spectrum of compound 1 (lithocarpinol B).
Fig. 6 is the NOESY spectrum of compound 1 (lithocarpinol B).
Fig. 7 is the HRESIMS spectrum of compound 1 (lithocarpinol B).
Fig. 8 is the CD spectrum of compound 1 (lithocarpinol B).
Fig. 9 is the ECD matched curve of compound 1 (lithocarpinol B).
Figure 10 is the UV spectrum of compound 1 (lithocarpinol B).
Figure 11 is the IR spectrum of compound 1 (lithocarpinol B).
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
Embodiment 1
(1) the deep-sea fungi Phomopsis lithocarpus FS508 of activation is linked into potato glucose liquid
(preparation method is culture medium：Potato is cleaned into peeling, being cut into size is about 1cm3Fritter, weigh 200g, be put into 1L water
It boils 20-30 minutes, is filtered with double gauze, take filtrate, sea salt 15g, glucose 20g are added in filtrate, water is added to supply 1L,
Sterilizing is made) in, 28 DEG C, 120rpm, seed liquor is made in culture 5d, and seed liquor is linked into rice with the inoculum concentration of 10%mL/g
(preparation method is solid fermentation culture medium：3g sea salt and 160g rice are weighed respectively, are added 200mL water, are stirred, sterilizing system
), stationary culture one month, is made the fermentation culture medium of Phomopsis lithocarpus FS508 at room temperature.
(2) fermentation culture medium that step (1) obtains is extracted with ethyl acetate, extracts 4 times, acetic acid ethyl acetate extract is through distilling
Medicinal extract is obtained after concentration, is that 85% methanol aqueous solution dissolves medicinal extract with suitable volume fraction, then three times with petroleum ether extraction,
To remove low polar fatty acid material, remaining methanol aqueous solution part distillation and concentration obtains brownish black medicinal extract.
(3) the brownish black medicinal extract that step (2) obtains is crossed into normal phase silicagel column, first uses petroleum ether-ethyl acetate from 10:1,7:
1,9:2,3:1,2:1,1:1,1:2v/v gradient elution, then eluted with ethyl acetate, finally use methylene chloride：Methanol=5:1,1:
1v/v elution；Medicinal extract is collected by petroleum ether：Ethyl acetate volume ratio 3:The fraction F6 of 1 elution, component F6 is through Sephadex LH-
20 columns, with methylene chloride：Methanol=1:1v/v collects TLC thin-layer chromatography as eluant, eluent with petroleum ether：Ethyl acetate=2:
1v/v be unfolded Rf=3.0/3.8 component, normal-phase silica gel column chromatography is then crossed, with petroleum ether-ethyl acetate from 6:1,3:1,
2:1,1:1,1:2,0:1v/v gradient elution collects TLC thin-layer chromatography with methylene chloride：Methanol=20:Rf=is unfolded to obtain in 1v/v
3.3/3.6 component, then separated the component with HPLC prepare entirely (the full chromatographic condition for preparing HPLC is：YMC ODS-A column, stream
Dynamic is mutually volume ratio 85:15 methanol/water, flow velocity 8mL/min), eluting fraction is collected at retention time 14.8min, most
Prepare HPLC with half afterwards to purify the fraction (chromatographic condition for partly preparing HPLC is：YMC-pack ODS-A/AQ column, mobile phase are
Volume ratio 82:18 acetonitrile/water, flow velocity 3mL/min, retention time 9.4min), obtain 1 (compound of compound
lithocarpinol B)。
For compound 1 through mass spectrum and spectral analysis of the nuclear magnetic resonance, Structural Identification is as follows：
As shown in figs. 1-11, Fig. 1 is compound 11H-NMR spectrum；Fig. 2 is compound 113C-NMR spectrum；Fig. 3 is chemical combination
The hsqc spectrum of object 1；Fig. 4 is compound 11H-1H COSY spectrum；Fig. 5 is the HMBC spectrum of compound 1；Fig. 6 is compound 1
NOESY spectrum；Fig. 7 is the HRESIMS spectrum of compound 1；Fig. 8 is the CD spectrum of compound 1；Fig. 9 is the ECD matched curve of compound 1；
Figure 10 is the UV spectrum of compound 1；Figure 11 is the IR spectrum of compound.
Compound 1 has following physical and chemical and spectral characteristic：White solid powder；
UV(MeOH)λmax(logε)208.8(3.86),264.8(3.09)nm；CD(0.16mg/mL,MeOH)λmax(Δε)214(–
42.07),229(–5.68),246(–1.98),277(+6.34)nm；IRνmax 3360,2943,1645,1479,1286cm-1。
1H and13C NMR data；HRESIMS m/z[M+Na]+447.1778(calcd for C25H28NaO6,
447.1778)。1H-NMR(500MHz,CDCl3)δ:9.82 (1H, s, H-13), 7.54 (1H, d, J=8.4Hz, H-5), 7.07
(1H, s, H-6'), 6.87 (1H, d, J=8.4Hz, H-4), 6.68 (1H, d, J=1.8Hz, H-4'), 4.91 (1H, s, H-
11b), 4.76 (1H, s, H-11a), 4.41 (1H, dd, J=11.0,2.0Hz, H-8a'), 3.79 (1H, dd, J=9.3,
2.0Hz, H-9'), 3.70 (1H, m, H-8b'), 3.59 (1H, m, H-8), 3.23 (1H, dd, J=15.3,9.3Hz, H-7a),
2.94 (1H, dd, J=15.3,7.8Hz, H-7b), 2.24 (1H, s, H-7'), 1.62 (3H, s, H-10), 0.99 (3H, s, H-
11'),0.75(3H,s,H-12')。13C-NMR(125MHz,CDCl3)δ:196.6(C-13),162.5(C-3),151.3(C-
1),144.5(C-3'),144.2(C-9),138.4(C-2'),135.7(C-1'),134.7(C-6),134.4(C-5),130.7
(C-5'),120.3(C-6'),118.3(C-4),117.7(C-4'),117.1(C-2),114.4(C-11),84.5(C-12),
80.2(C-9'),70.3(C-10'),65.4(C-8'),57.8(C-8),34.5(C-7),27.5(C-11'),23.9(C-
12'),23.5(C-10),21.1(C-7').CD modal data shows that the compound shows positive cotton effect in 277nm,
214,229,246nm shows negative cotton effect, and the ECD matched curve of the 8S with calculating, 12R, 9'S are consistent (Fig. 9).Cause
This, can determine that the absolute configuration of the compound 1 is 8S, 12R, 9'S.By literature search, which is new skeleton compounds
Object belongs to 2,3-dihydro-1H-indene class noval chemical compound, is named as lithocarpinol B, in specific structure such as Formulas I
It is shown.
Shown in the structural formula such as following formula (I) of isolated compound lithocarpinol B：
Embodiment 2
Using filter paper enzyme measurement compound lithocarpinol B to the antibacterial activity of aspergillus flavus.
It is 1.0 × 10 that the aspergillus flavus of logarithmic growth phase, which is assigned to spore count with physiological saline,6The spore suspension of a/mL, will
Compound lithocarpinol B and positive control medicine nystatin be diluted to respectively 5 μ g/mL, 10 μ g/mL, 20 μ g/mL,
40 μ g/mL, 60 μ g/mL, 80 μ g/mL, 100 μ g/mL concentration.It takes spore suspension 1mL in culture dish, pours into PDA culture medium,
It is uniformly mixed, places 6mm in culture dish containing bacterium, 5 μ L of sample diluting liquid is added dropwise respectively on filter paper, blank control is added dropwise
5 μ L of DMSO, makees positive control with nystatin, and plate is placed in 26 DEG C of insulating box culture 72h, observes the suppression around filter paper
Bacterium circle, to there is institute's minimum concentration with sample of inhibition zone as minimum inhibitory concentration (MIC value) around filter paper.
Experimental result：Compound lithocarpinol B is 20 μ g/mL, positive control medicine system to the MIC value of aspergillus flavus
Moldin is 15 μ g/mL to aspergillus flavus MIC value.The result shows that：The compound of the present invention lithocarpinol B has aspergillus flavus
There is apparent inhibitory activity, can be used to prepare antifungal drug.Therefore, the present invention provides candidates for antimycotic new drug development
Object is closed, provides scientific basis for development and utilization marine fungi resource.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (10)
1. compound lithocarpinol B, which is characterized in that shown in structure such as formula (I)：
2. a kind of preparation method of compound lithocarpinol B described in claim 1, which is characterized in that the change
Closing object lithocarpinol B is separated from the fermentation culture medium of deep-sea fungi Phomopsis lithocarpus FS508
It is prepared.
3. preparation method according to claim 2, which is characterized in that specifically include following steps：
(1) fermented and cultured is extracted with ethyl acetate in the fermentation culture medium for preparing Phomopsis lithocarpus FS508
Acetic acid ethyl acetate extract is obtained medicinal extract by object after distillation and concentration；Medicinal extract is dissolved with methanol aqueous solution, is then extracted with petroleum ether
It takes, extracts remaining methanol aqueous solution part distillation and concentration and obtain brownish black medicinal extract；
(2) brownish black medicinal extract is crossed into normal phase silicagel column, with petroleum ether-ethyl acetate from 10:1,7:1,9:2,3:1,2:1,1:1,
1:2v/v gradient elution collects petroleum ether：Ethyl acetate volume ratio 3:The fraction F6 of 1 elution, component F6 is through Sephadex LH-
20 columns, with methylene chloride：Methanol=1:1v/v collects TLC thin-layer chromatography as eluant, eluent with petroleum ether：Ethyl acetate=2:
1v/v be unfolded Rf=3.0/3.8 component, normal-phase silica gel column chromatography is then crossed, with petroleum ether-ethyl acetate from 6:1,3:1,
2:1,1:1,1:2,0:1v/v gradient elution collects TLC thin-layer chromatography with methylene chloride：Methanol=20:Rf=is unfolded to obtain in 1v/v
3.3/3.6 component, then purified to obtain compound lithocarpinol B with HPLC.
4. preparation method according to claim 3, which is characterized in that the HPLC purifying is with full preparation HPLC separation
The component, the chromatographic condition for preparing HPLC entirely are：YMC ODS-A column, mobile phase are volume ratio 85:15 methanol/water, flow velocity are
8mL/min collects eluting fraction at retention time 14.8min, finally purifies the component with half preparation HPLC, half preparation HPLC
Chromatographic condition be：YMC-pack ODS-A/AQ column, mobile phase are volume ratio 82:18 acetonitrile/water, flow velocity 3mL/min,
Retention time 9.4min obtains compound lithocarpinol B.
5. preparation method according to claim 3, which is characterized in that the preparation Phomopsis lithocarpus
The fermentation culture medium of FS508 is that the Phomopsis lithocarpus FS508 strain of activation is linked into potato glucose
In fluid nutrient medium, 28 DEG C, 120rpm, seed liquor is made in culture 5d, and seed liquor is linked into greatly with the inoculum concentration of 10%mL/g
In rice solid fermentation culture medium, stationary culture one month, is made fermentation culture medium, the potato glucose liquid training at room temperature
Feeding base obtains in the following manner：Potato is cleaned into peeling, being cut into size is about 1cm3Fritter, weigh 200g, be put into 1L
It is boiled in water 20~30 minutes, is filtered with double gauze, take filtrate, sea salt 15g, glucose 20g are added in filtrate, water is added to mend
Sufficient 1L, sterilizing are made；The rice solid fermentation culture medium obtains in the following manner：It weighs 3g sea salt respectively and 160g is big
Rice, adds 200mL water, is stirred, and sterilizing is made.
6. deep-sea fungi Phomopsis lithocarpus FS508 is preparing compound described in claim 1
Application in lithocarpinol B.
7. compound lithocarpinol B described in claim 1 is preparing the application in antifungal drug.
8. application according to claim 7, which is characterized in that the antifungal drug is the drug of aspergillus flavus resisting.
9. a kind of antifungal drug, which is characterized in that contain compound lithocarpinol B conduct described in claim 1
Effective component.
10. antifungal drug according to claim 9, which is characterized in that the antifungal drug is aspergillus flavus resisting
Drug.
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| CN109503623A (en) * | 2018-11-30 | 2019-03-22 | 广东省微生物研究所（广东省微生物分析检测中心） | Guanacastane class compound and preparation method thereof and the application in preparation antibacterials |
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| CN112852647A (en) * | 2021-02-23 | 2021-05-28 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Overexpression vector suitable for phomopsis FS508 and construction method and application thereof |
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| CN109232513B (en) * | 2018-09-18 | 2020-06-16 | 广东省微生物研究所（广东省微生物分析检测中心） | Compound litocarpinols, preparation method thereof and application thereof in preparation of antitumor drugs |
| CN109503623A (en) * | 2018-11-30 | 2019-03-22 | 广东省微生物研究所（广东省微生物分析检测中心） | Guanacastane class compound and preparation method thereof and the application in preparation antibacterials |
| CN109503623B (en) * | 2018-11-30 | 2020-04-21 | 广东省微生物研究所（广东省微生物分析检测中心） | Guanactastane compound, preparation method thereof and application thereof in preparation of antibacterial drugs |
| CN110862371A (en) * | 2019-11-19 | 2020-03-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Polycyclic polyketone compound, preparation method thereof and application thereof in preparation of antibacterial drugs |
| CN110862371B (en) * | 2019-11-19 | 2022-10-04 | 广东省微生物研究所（广东省微生物分析检测中心） | Polycyclic polyketone compound, preparation method thereof and application thereof in preparation of antibacterial drugs |
| CN112592350A (en) * | 2020-12-18 | 2021-04-02 | 广东省微生物研究所（广东省微生物分析检测中心） | Polyketide lithocarpin E-G and preparation method and application thereof |
| CN112852647A (en) * | 2021-02-23 | 2021-05-28 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Overexpression vector suitable for phomopsis FS508 and construction method and application thereof |
| CN112852647B (en) * | 2021-02-23 | 2022-06-07 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Overexpression vector suitable for phomopsis FS508 and construction method and application thereof |
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