CN116926143A - Aromatic polyketone compound and preparation method and application thereof - Google Patents
Aromatic polyketone compound and preparation method and application thereof Download PDFInfo
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- 229920001470 polyketone Polymers 0.000 title claims abstract description 20
- 125000003118 aryl group Chemical group 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 150000001875 compounds Chemical class 0.000 title claims description 14
- 229930014544 aromatic polyketide Natural products 0.000 claims abstract description 28
- 125000003822 aromatic polyketide group Chemical group 0.000 claims abstract description 27
- 241000233866 Fungi Species 0.000 claims abstract description 24
- 241000209140 Triticum Species 0.000 claims abstract description 24
- 235000021307 Triticum Nutrition 0.000 claims abstract description 24
- 206010039509 Scab Diseases 0.000 claims abstract description 19
- 241000238631 Hexapoda Species 0.000 claims abstract description 16
- 239000000575 pesticide Substances 0.000 claims abstract description 13
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 claims abstract description 12
- 230000000968 intestinal effect Effects 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 51
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 39
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 32
- 238000000855 fermentation Methods 0.000 claims description 24
- 239000003480 eluent Substances 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 19
- 239000003208 petroleum Substances 0.000 claims description 19
- 239000000741 silica gel Substances 0.000 claims description 19
- 229910002027 silica gel Inorganic materials 0.000 claims description 19
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 18
- 241001093951 Ailanthus altissima Species 0.000 claims description 17
- 239000000287 crude extract Substances 0.000 claims description 15
- 239000011347 resin Substances 0.000 claims description 15
- 229920005989 resin Polymers 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 14
- 238000010898 silica gel chromatography Methods 0.000 claims description 14
- 238000001179 sorption measurement Methods 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 5
- 241000189150 Nigrospora Species 0.000 claims description 4
- 238000013375 chromatographic separation Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 3
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- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
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- 238000010828 elution Methods 0.000 claims description 3
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 238000004080 punching Methods 0.000 claims description 3
- 238000000638 solvent extraction Methods 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 210000001835 viscera Anatomy 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 2
- 241000223195 Fusarium graminearum Species 0.000 abstract description 17
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 11
- 239000005747 Chlorothalonil Substances 0.000 abstract description 8
- CRQQGFGUEAVUIL-UHFFFAOYSA-N chlorothalonil Chemical compound ClC1=C(Cl)C(C#N)=C(Cl)C(C#N)=C1Cl CRQQGFGUEAVUIL-UHFFFAOYSA-N 0.000 abstract description 8
- 230000002401 inhibitory effect Effects 0.000 abstract description 6
- 229930000044 secondary metabolite Natural products 0.000 abstract description 4
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 2
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- 230000000877 morphologic effect Effects 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241001557905 Nigrospora sp. Species 0.000 description 2
- -1 aromatic polyketide compound Chemical class 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000005100 correlation spectroscopy Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000035784 germination Effects 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
- 150000002611 lead compounds Chemical class 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000004763 spore germination Effects 0.000 description 2
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000368696 Nigrospora oryzae Species 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/20—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom three- or four-membered rings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
- C07D303/02—Compounds containing oxirane rings
- C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
- C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
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- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
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- C07B2200/07—Optical isomers
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pest Control & Pesticides (AREA)
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Abstract
The invention belongs to the field of microorganism secondary metabolites, and particularly discloses an aromatic polyketide C which is separated from the secondary metabolites of insect intestinal fungi and obtained by a preparation method and application thereof 12 H 12 O 4 The aromatic polyketone compound C 12 H 12 O 4 Has good antibacterial activity to Fusarium graminearum, the antibacterial rate reaches 100% at the final concentration of 100 mug/mL, the antibacterial activity is equivalent to that of control agent chlorothalonil in the aspect of inhibiting hypha growth, and the antibacterial activity to spores of Fusarium graminearum is strongActing; therefore, the aromatic polyketone compound C prepared by the invention 12 H 12 O 4 Can be used for preventing and treating wheat scab or preparing pesticides for preventing and treating wheat scab.
Description
Technical Field
The invention relates to the field of microorganism secondary metabolites, in particular to an aromatic polyketide compound, a preparation method and application thereof.
Background
Wheat is one of three grains in China, and has a critical influence on the grain production safety in China. Wheat scab (Fusarium Head Blight) caused by fusarium graminearum (Fusarium graminearum) is one of the main factors responsible for the massive reduction of wheat yield in China. Wheat scab seriously jeopardizes the world's wheat production safety, and has continuously occurred and is widely prevalent in China and other countries of the world in recent decades. Statistics show that the number of the severe outbreaks of wheat scab in China reaches 10 since 2000, and the incidence area exceeds 333 ten thousand hm 2 Of which 3 are pandemics.
The method for preventing and controlling wheat scab by adopting resistance breeding is the most economical, safe and effective method, but the current resistance resource is seriously deficient, so that the aim of fundamentally preventing and controlling wheat scab is difficult to achieve. Biological control accords with the environmental protection theme, but the biological control is difficult to realize commercial production for controlling wheat scab at the present stage due to the limiting factors of the biological control. At present, chemical pesticides are mostly relied on to control wheat scab in large-scale wheat production, and the drug resistance of fusarium graminearum to the traditional chemical pesticides is enhanced year by year, so that the local control effect is reduced continuously, and the diseases are difficult to effectively control for a long time. Therefore, the development of novel pesticides for controlling wheat scab caused by fusarium graminearum has been hampered to alleviate the problems of resistance to chemicals and pollution, etc. of the conventional chemical pesticides currently used.
Disclosure of Invention
In order to solve the technical problems, the invention provides an aromatic polyketone compound, and a preparation method and application thereof.
In order to achieve the above purpose, the invention is implemented according to the following technical scheme:
the first object of the present invention is to provide a method for preparing an aromatic polyketone compound, comprising the steps of:
s1, separating a strain of fungus Nigrospora sp from insect ailanthus altissima intestinal flora, wherein the strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40671, the preservation date of 2023.6.25 and the preservation address of North Chen Xili No. 1 in the Korean region of Beijing city;
s2, performing 24L fermentation treatment on the black spore fungus SN56127, and obtaining a black spore fungus SN56127 fermentation crude extract by adopting a macroporous adsorption resin adsorption and solvent extraction technology;
s3, separating the fermented crude extract of the black spore fungus SN56127 by using a silica gel and gel column chromatography technology to obtain the aromatic polyketide.
Further, the step S1 specifically includes:
firstly, cleaning the surface of an insect ailanthus altissima by using 95% ethanol and sterile water, cutting open an ailanthus altissima mouth device and a tail part of the insect ailanthus altissima by using a sterilized scalpel, slightly taking out an intestinal tract from the head part of the insect ailanthus altissima mouth device and the tail part of the insect ailanthus altissima mouth device by using sterilized forceps, removing viscera and accessories, collecting the content of the intestinal tract, adding a proper amount of phosphoric acid buffer solution into the content of the intestinal tract to dilute the content, uniformly coating the diluted content on a PDA (personal digital assistant) culture medium, and culturing the culture medium in a culture box at 28 ℃ for 48 hours; single colonies of different forms were picked using an inoculating needle, and cultivation was repeated to obtain pure strain SN56127.
Further, the step S2 specifically includes:
s21, adding 5mL of PDB culture solution into a 25mL test tube, and sterilizing for 30min at 121 ℃; inoculating the black spore filamentous fungus SN56127 stored in a refrigerator at-80 ℃ into a PDA culture medium, and culturing at constant temperature of 25 ℃ for 7d; punching holes on the edge of the bacterial colony by using a 5mm puncher when the diameter of the bacterial colony grows to about 7cm, inoculating bacterial cakes into 25mL test tubes to serve as primary liquid seeds, placing all the test tubes into a shaking table after inoculation, shake-culturing at 180r/min and 25 ℃ for 48 hours, and taking out the bacterial liquid when the bacterial liquid is turbid;
s22, transferring all the fermented primary seeds into 250mL triangular flasks to serve as secondary liquid seeds, adding 50mL of PDB culture medium into each triangular flask, performing wet heat sterilization at 121 ℃ for 30min,180r/min, performing shake culture at 25 ℃ for 48h, and taking out when bacterial liquid is turbid and bacterial blocks are formed;
s23, transferring all the fermented secondary seeds into 2L triangular flasks, adding 400mL of PDB culture medium and 16g of macroporous adsorption resin into each triangular flask, performing wet heat sterilization at 121 ℃ for 30min,180r/min, performing shake culture at 25 ℃ for 7d, and performing co-fermentation for 24L;
s24, after the whole fermentation is finished, obtaining 24L of fermentation liquor of the black spore filamentous fungi SN56127, flushing the fermentation liquor of the black spore filamentous fungi SN56127 for a plurality of times by using distilled water, completely removing thalli and a culture medium in the fermentation liquor of the black spore filamentous fungi SN56127, collecting macroporous adsorption resin, collecting the macroporous adsorption resin in a bottle into a tray, putting the tray into a baking oven, and drying at the temperature of 30 ℃; drying the resin, placing into a 2L separating funnel, pouring a proper amount of methanol for eluting, changing methanol every 4 hours of soaking, soaking and eluting for 5 times, wherein one time is soaked overnight, and collecting and combining the eluates; concentrating the eluent under reduced pressure to obtain a crude fermentation extract; and dissolving the fermented crude extract by using methanol, adding distilled water and dichloromethane with the same volume for extraction, extracting for 5 times, collecting a dichloromethane phase, and concentrating under reduced pressure to obtain a dichloromethane crude extract.
Further, the step S3 specifically includes:
s31, using a petroleum ether-ethyl acetate system as an eluent for primary silica gel column chromatography to perform gradient elution on a dichloromethane crude extract, wherein the gradient eluent of a normal phase silica gel column is respectively selected from petroleum ether, petroleum ether and ethyl acetate with volume ratios of 100:1, 100:2, 100:4, 100:8, 100:16, 1:1 and ethyl acetate, and each mobile phase is eluted by 0.6L; after the first-stage silica gel column chromatography is finished, the fractions are combined into A, B according to the polarity 1 、B 2 And B 3 Four components;
s32, selecting petroleum ether and ethyl acetate with the volume ratio of 100:8 as gradient eluent of a normal phase silica gel column, and performing column chromatography on B 3 Separating the components by first silica gel column chromatography to obtain B 31 The components are selected from petroleum ether and ethyl acetate with the volume ratio of 100: 3. 100:8 and 9:1 gradient eluent pair B as normal phase silica gel column 31 Separating the components by a second silica gel column chromatography to obtain B 32 The components are petroleum ether, methylene dichloride and methanol with the volume ratio of 2:1:1 gradient eluent pair as normal phase silica gel columnB 32 The components are subjected to a third gel column chromatographic separation to obtain B 33 The components are dichloromethane and methanol with the volume ratio of 100:0.04 gradient eluent pair B as normal phase silica gel column 33 The components are subjected to fourth silica gel column chromatography separation to obtain an aromatic polyketone compound C 12 H 12 O 4 。
A second object of the present invention is to provide an aromatic polyketone compound C prepared by the above method 12 H 12 O 4 。
A third object of the present invention is to provide an aromatic polyketone compound C 12 H 12 O 4 The application in preventing and controlling wheat scab.
A fourth object of the present invention is to provide an aromatic polyketone compound C 12 H 12 O 4 The application in preparing pesticide for preventing and treating wheat scab.
Compared with the prior art, the invention separates and obtains the aromatic polyketide C from the secondary metabolite of the insect intestinal fungi 12 H 12 O 4 The aromatic polyketone compound C 12 H 12 O 4 The composition has good antibacterial activity on Fusarium graminearum, the antibacterial rate reaches 100% at the final concentration of the compound of 100 mug/mL, the composition is equivalent to control agent chlorothalonil in the aspect of inhibiting hypha growth, and the composition has strong germination inhibiting effect on spores of Fusarium graminearum; therefore, the aromatic polyketone compound C prepared by the invention 12 H 12 O 4 Can be used for preventing and treating wheat scab or preparing pesticides for preventing and treating wheat scab, can effectively solve the problem of drug resistance caused by preventing and treating wheat scab by adopting traditional chemical pesticides in the current stage of China, can reduce the problems of ecological environment pollution, poisoning of pesticides of human beings and animals and the like, and is beneficial to the safe production of wheat in China.
Drawings
FIG. 1 is a morphological diagram of the fungus, the genus Heidellum SN56127.
FIG. 2 shows an aromatic polyketide C 12 H 12 O 4 Is a high resolution mass spectrum of (c).
FIG. 3 is a schematic view ofAromatic polyketone compound C 12 H 12 O 4 Nuclear magnetic hydrogen spectrum of (2).
FIG. 4 shows an aromatic polyketide C 12 H 12 O 4 Nuclear magnetic carbon spectrum of (a).
FIG. 5 shows an aromatic polyketide C 12 H 12 O 4 Is a HSQC spectrum of (C).
FIG. 6 is an aromatic polyketide C 12 H 12 O 4 Is a COSY spectrum of (C).
FIG. 7 is an aromatic polyketide C 12 H 12 O 4 HMBC spectra of (a).
FIG. 8 is an aromatic polyketide C 12 H 12 O 4 Single crystal diffraction results of (2).
FIG. 9 is an aromatic polyketide C 12 H 12 O 4 Antibacterial effect on growth of Fusarium graminearum hypha.
FIG. 10 shows an aromatic polyketide C 12 H 12 O 4 Effects on Fusarium graminearum spore germination.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
Example 1
Firstly, cleaning the surface of an insect ailanthus altissima by using 95% ethanol and sterile water, cutting open an ailanthus altissima mouth device and a tail part of the insect ailanthus altissima by using a sterilized scalpel, slightly taking out an intestinal tract from the head part of the insect ailanthus altissima mouth device and the tail part of the insect ailanthus altissima mouth device by using sterilized forceps, removing viscera and accessories, collecting the content of the intestinal tract, adding a proper amount of phosphoric acid buffer solution into the content of the intestinal tract to dilute the content, uniformly coating the diluted content on a PDA (personal digital assistant) culture medium, and culturing the culture medium in a culture box at 28 ℃ for 48 hours; single colonies of different forms were picked using an inoculating needle, and the culture was repeated to obtain pure strain SN56127, which was preliminarily estimated to be a Nigrospora fungus (Nigrospora sp.) by morphological characteristics and molecular biological analysis, and the Nigrospora fungus SN56127 had the following characteristics:
a) Morphological characteristics: the colony is white velvet-shaped, hypha grows luxuriantly, the colony is opaque, and the back surface of the colony is light yellow in the later growth period, as shown in figure 1.
Phylogenetic characteristics: phylogenetic comparison analysis is carried out by amplifying rDNA-ITS sequences, and the result shows that the ITS-rDNA sequences of the strain are 100% similar to those of a standard strain Nigrospora oryzae strain CEF-002 (GenBank accession No. KF 998977.1). The results of gene sequencing were as follows:
GGGCTTCGAGTCTCCACTCCCATACCCATGTGACTTATCTCTTTGTTGCCTCGGCGCAAGCTACCCGGGACCCAGCGCCCCGGGCGGCCCGCCGGCGGACAAACCAAACTCTTGTTATCTTAGTTGATTATCTGAGCGTCTTATTTAATAAGTCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCATTAGTATTCTAGTGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTAAGCACAGCTTACTGTTGGGACTCTACGGCCTCCGTAGTTCCCCAAAGCGATTGGCGGAGTGGCAGTAGTCCTCTGAGCGTAGTAATTTTTTATCTCGCTTTTGTTAGGTGCTGCCCCCCCGGCCGTTAAACCCCCCCAATTTTTTCTGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAAAGCGGAGGAA。
nigrospora sp. Deposited with China general microbiological culture Collection center (CGMCC) with accession number of CGMCC No.40671, date of deposit of 2023.6.25 and deposited address of Beijing Chaoyang area North Chen Xiu No. 1 and 3.
Example 2
Performing 24L fermentation treatment on the black spore fungus SN56127 obtained in the example 1, and obtaining a black spore fungus SN56127 fermentation crude extract by using a macroporous adsorption resin adsorption and solvent extraction technology; then separating the fermented crude extract of the black spore fungus SN56127 by using a silica gel and gel column chromatography technology to obtain the aromatic polyketide, wherein the specific process is as follows:
1) 5mL of PDB culture solution is added into a 25mL test tube, and sterilization is carried out for 30min at 121 ℃; inoculating the black spore filamentous fungi SN-56127 stored in a refrigerator at the temperature of minus 80 ℃ into a PDA culture medium, culturing at the constant temperature of 25 ℃ for 7 days, punching holes at the edges of colonies by a 5mm puncher when the diameters of the colonies grow to about 7cm, inoculating a bacterial cake into a 25mL test tube to serve as primary liquid seeds, and taking 3 bottles of test tubes not inoculated as blank control; after inoculation, all test tubes are put into a shaking table, 180r/min and shake culture is carried out at 25 ℃ for 48 hours, and the test tubes are taken out when bacterial liquid is turbid;
2) Transferring all the fermented primary seeds into 250mL triangular flasks to serve as secondary liquid seeds, adding 50mL of PDB culture medium into each triangular flask, performing wet heat sterilization at 121 ℃ for 30min, taking 3 triangular flasks as blank control, performing shake culture at 180r/min at 25 ℃ for 48h, and taking out when bacterial liquid is turbid and bacterial blocks are formed;
3) Transferring all the fermented secondary seeds into 2L triangular flasks, adding 400mLPDB culture medium and 16g macroporous adsorbent resin (XAD-16) into each triangular flask, performing wet heat sterilization at 121 ℃ for 30min, performing shake culture at 25 ℃ for 7d with 3 triangular flasks not inoculated as blank control at 180r/min, and performing co-fermentation for 24L;
4) After the whole fermentation is finished, obtaining 24L of fermentation liquor of the black spore filamentous fungi SN56127, flushing the fermentation liquor of the black spore filamentous fungi SN56127 for a plurality of times by using distilled water, completely removing thalli and a culture medium in the fermentation liquor of the black spore filamentous fungi SN56127, collecting macroporous adsorption resin, completely gathering the macroporous adsorption resin in a bottle into a tray, putting the tray into a baking oven, and drying at the temperature of 30 ℃; drying the resin, placing into a 2L separating funnel, pouring a proper amount of methanol for eluting, changing methanol every 4 hours of soaking, soaking and eluting for 5 times, wherein one time is soaked overnight, and collecting and combining the eluates; concentrating the eluent under reduced pressure to obtain a crude fermentation extract; dissolving the fermented crude extract by using methanol, adding 300ml of distilled water and 300ml of dichloromethane for extraction, extracting for 5 times, collecting dichloromethane phase, and concentrating under reduced pressure to obtain 15.44g of dichloromethane crude extract;
5) Using petroleum ether-ethyl acetate system as eluent of primary silica gel column chromatography to perform gradient elution on dichloromethane crude extract, respectively selecting petroleum ether, petroleum ether and ethyl acetate with volume ratios of 100:1, 100:2, 100:4, 100:8, 100:16, 1:1 and ethyl acetate as gradient eluent of normal phase silica gel column, and eluting with 0.6L of mobile phase each time; after the primary silica gel column chromatography is finished, the fractions are combined according to the polarity to obtain 0.1189gA components and 0.0284g B 1 Component 1.1919g B 2 Component (A) and 6.1201g B 3 A component (C);
6) The petroleum ether and ethyl acetate with the volume ratio of 100:8 are selected as gradient eluent of a normal phase silica gel column, and the ratio of petroleum ether to ethyl acetate is equal to B 3 Separating the components by first silica gel column chromatography to obtain B 31 The components are selected from petroleum ether and ethyl acetate with the volume ratio of 100: 3. 100:8 and 9:1 gradient eluent pair B as normal phase silica gel column 31 Separating the components by a second silica gel column chromatography to obtain B 32 The components are petroleum ether, methylene dichloride and methanol with the volume ratio of 2:1:1 gradient eluent pair B as normal phase silica gel column 32 The components are subjected to a third gel column chromatographic separation to obtain B 33 The components are dichloromethane and methanol with the volume ratio of 100:0.04 gradient eluent pair B as normal phase silica gel column 33 The components are subjected to a fourth silica gel column chromatographic separation to obtain an aromatic polyketone compound;
the structure of the aromatic polyketide was identified, and the high resolution mass spectrum of the aromatic polyketide was shown in FIG. 2, according to the high resolution HRESIMS (m/z 221.0811[ M+H ]] + Calculated value C 12 H 13 O 4 221.0814) to determine its molecular formula as C 12 H 12 O 4 The unsaturation was 7. According to 1 H NMR spectrum, 13 The results of the analysis of the data of C NMR spectrum, HSQC spectrum, COSY spectrum and HMBC spectrum and the verification of single crystal diffraction are shown in Table 1 and FIG. 3 to FIG. 8.
TABLE 1
From FIGS. 3 to 8, it can be identified that the aromatic polyketide has the structure of (E) -1- (2, 4-dihydroxyphenyl) -3- ((2S, 3S) -3-methylmercxiran-2-yl) prop-2-en-1-one, and the aromatic polyketide has the structural formula:the two-dimensional correlation of the aromatic polyketide is as follows:
the structure of the compound is searched by SCI Finder, no related report is found, and the compound is determined to be a novel compound.
Example 3
Determination of aromatic polyketide C obtained in example 2 by hypha growth Rate method 12 H 12 O 4 Antibacterial activity of Fusarium graminearum, treating with chlorothalonil as positive control, and calculating EC of compound and chlorothalonil for inhibiting growth of Fusarium graminearum mycelium by mycelium growth inhibition test 50 Value, aromatic polyketide C 12 H 12 O 4 The final concentrations of chlorothalonil and chlorothalonil were set to 6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL and 100. Mu.g/mL for a total of 5 gradient concentrations, and the results are shown in FIG. 9 and Table 2.
TABLE 2
The result shows that the antibacterial rate of the compound to Fusarium graminearum reaches 100% at the concentration of 100 mug/mL, and EC 50 EC with value of 24.37 mug/mL, chlorothalonil 50 The value was 27.52. Mu.g/mL.
Example 4
The aromatic polyketide C is tested by adopting spore germination inhibition 12 H 12 O 4 Inhibition effect on fusarium graminearum spores, aromatic polyketide C 12 H 12 O 4 The final concentrations were set to 5 gradient concentrations of 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL and 10. Mu.g/mL, and the EC was calculated 50 The values and results are shown in fig. 10 and table 3.
TABLE 3 Table 3
The result shows that the aromatic polyketone compound C 12 H 12 O 4 The inhibition rate of fusarium graminearum spores reaches 100% at the concentration of 10 mug/mL, and EC 50 The value was 5.10. Mu.g/mL.
Taken together, it is shown that the aromatic polyketide C 12 H 12 O 4 The composition has good antibacterial activity on Fusarium graminearum, the antibacterial rate reaches 100% at the final concentration of 100 mug/mL, the composition is equivalent to control agent chlorothalonil in the aspect of inhibiting hypha growth, and the composition has strong germination inhibiting effect on spores of Fusarium graminearum. The aromatic polyketone compound C 12 H 12 O 4 The potential value of the novel environment-friendly pesticide lead compound can be developed, and the novel environment-friendly pesticide lead compound can be used for preventing and treating wheat scab or preparing pesticides for preventing and treating wheat scab.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (7)
1. The preparation method of the aromatic polyketone compound is characterized by comprising the following steps of:
s1, separating a strain of fungus Nigrospora from insect ailanthus altissima intestinal flora, wherein the strain is preserved in the China general microbiological culture Collection center (China Committee) with the preservation number: CGMCCNO.40671 with a preservation date of 2023.6.25 and a preservation address of Beijing Chaoyang area North Chenxi No. 1 and 3;
s2, performing 24L fermentation treatment on the black spore fungus SN56127, and obtaining a black spore fungus SN56127 fermentation crude extract by adopting a macroporous adsorption resin adsorption and solvent extraction technology;
s3, separating the fermented crude extract of the black spore fungus SN56127 by using a silica gel and gel column chromatography technology to obtain the aromatic polyketide.
2. The method for preparing aromatic polyketone compounds according to claim 1, wherein the step S1 specifically comprises:
firstly, cleaning the surface of an insect ailanthus altissima by using 95% ethanol and sterile water, cutting open an ailanthus altissima mouth device and a tail part of the insect ailanthus altissima by using a sterilized scalpel, slightly taking out an intestinal tract from the head part of the insect ailanthus altissima mouth device and the tail part of the insect ailanthus altissima mouth device by using sterilized forceps, removing viscera and accessories, collecting the content of the intestinal tract, adding a proper amount of phosphoric acid buffer solution into the content of the intestinal tract to dilute the content, uniformly coating the diluted content on a PDA (personal digital assistant) culture medium, and culturing the culture medium in a culture box at 28 ℃ for 48 hours; single colonies of different forms were picked using an inoculating needle, and cultivation was repeated to obtain pure strain SN56127.
3. The method for preparing aromatic polyketone compounds according to claim 1, wherein the step S2 specifically comprises:
s21, adding 5mLPDB culture solution into a 25mL test tube, and sterilizing for 30min at 121 ℃; inoculating the black spore filamentous fungus SN56127 stored in a refrigerator at-80 ℃ into a PDA culture medium, and culturing at constant temperature of 25 ℃ for 7d; punching holes on the edge of the bacterial colony by using a 5mm puncher when the diameter of the bacterial colony grows to about 7cm, inoculating bacterial cakes into 25mL test tubes to serve as primary liquid seeds, placing all the test tubes into a shaking table after inoculation, shake-culturing at 180r/min and 25 ℃ for 48 hours, and taking out the bacterial liquid when the bacterial liquid is turbid;
s22, transferring all the fermented primary seeds into 250mL triangular flasks to serve as secondary liquid seeds, adding 50mL of LPDB culture medium into each triangular flask, performing wet heat sterilization at 121 ℃ for 30min,180r/min, performing shake culture at 25 ℃ for 48h, and taking out when bacterial liquid is turbid and bacterial blocks are formed;
s23, transferring all the fermented secondary seeds into 2L triangular flasks, adding 400ml of LPDB culture medium and 16g of macroporous adsorption resin into each triangular flask, performing wet heat sterilization at 121 ℃ for 30min,180r/min, performing shake culture at 25 ℃ for 7d, and performing co-fermentation for 24L;
s24, after the whole fermentation is finished, obtaining 24L of fermentation liquor of the black spore filamentous fungi SN56127, flushing the fermentation liquor of the black spore filamentous fungi SN56127 for a plurality of times by using distilled water, completely removing thalli and a culture medium in the fermentation liquor of the black spore filamentous fungi SN56127, collecting macroporous adsorption resin, collecting the macroporous adsorption resin in a bottle into a tray, putting the tray into a baking oven, and drying at the temperature of 30 ℃; drying the resin, placing into a 2L separating funnel, pouring a proper amount of methanol for eluting, changing methanol every 4 hours of soaking, soaking and eluting for 5 times, wherein one time is soaked overnight, and collecting and combining the eluates; concentrating the eluent under reduced pressure to obtain a crude fermentation extract; and dissolving the fermented crude extract by using methanol, adding distilled water and dichloromethane with the same volume for extraction, extracting for 5 times, collecting a dichloromethane phase, and concentrating under reduced pressure to obtain a dichloromethane crude extract.
4. The method for preparing aromatic polyketide according to claim 3, wherein the step S3 specifically comprises:
s31, using a petroleum ether-ethyl acetate system as an eluent for primary silica gel column chromatography to perform gradient elution on a dichloromethane crude extract, wherein the gradient eluent of a normal phase silica gel column is respectively selected from petroleum ether, petroleum ether and ethyl acetate with volume ratios of 100:1, 100:2, 100:4, 100:8, 100:16, 1:1 and ethyl acetate, and each mobile phase is eluted by 0.6L; after the first-stage silica gel column chromatography is finished, the fractions are combined into A, B according to the polarity 1 、B 2 And B 3 Four components;
s32, selecting petroleum ether and ethyl acetate with the volume ratio of 100:8 as gradient eluent of a normal phase silica gel column, and performing column chromatography on B 3 Separating the components by first silica gel column chromatography to obtain B 31 The components are selected from petroleum ether and ethyl acetate with the volume ratio of 100: 3. 100:8 and 9:1 gradient eluent pair B as normal phase silica gel column 31 Separating the components by a second silica gel column chromatography to obtain B 32 The components are petroleum ether, methylene dichloride and methanol with the volume ratio of 2:1:1 gradient eluent pair B as normal phase silica gel column 32 The components are subjected to a third gel column chromatographic separation to obtain B 33 The components are dichloromethane and methanol with the volume ratio of 100:0.04 gradient eluent pair B as normal phase silica gel column 33 The components are subjected to fourth silica gel column chromatography separation to obtain an aromatic polyketone compound C 12 H 12 O 4 。
5. An aromatic polyketide C prepared by the method of any one of claims 1-4 12 H 12 O 4 。
6. A kind of rightThe aromatic polyketide C as claimed in claim 5 12 H 12 O 4 The application in preventing and controlling wheat scab.
7. An aromatic polyketide C as claimed in claim 5 12 H 12 O 4 The application in preparing pesticide for preventing and treating wheat scab.
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