CN113801006B - Actetrophenone A compound and preparation and application thereof - Google Patents

Actetrophenone A compound and preparation and application thereof Download PDF

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CN113801006B
CN113801006B CN202111141717.4A CN202111141717A CN113801006B CN 113801006 B CN113801006 B CN 113801006B CN 202111141717 A CN202111141717 A CN 202111141717A CN 113801006 B CN113801006 B CN 113801006B
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actetrophenone
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eluent
elution
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CN113801006A (en
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郭琳
谢则平
刘军鹏
徐琳琳
安振珍
李成强
李金权
王晨思
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Yantai Shuihetu Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
    • C07C49/703—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups
    • C07C49/747—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups containing six-membered aromatic rings
    • 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
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/06—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing keto or thioketo groups as part of a ring, e.g. cyclohexanone, quinone; Derivatives thereof, e.g. ketals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/78—Separation; Purification; Stabilisation; Use of additives
    • C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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    • 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
    • C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
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    • C07C2603/00—Systems containing at least three condensed rings
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    • C07C2603/40—Ortho- or ortho- and peri-condensed systems containing four condensed rings
    • C07C2603/42—Ortho- or ortho- and peri-condensed systems containing four condensed rings containing only six-membered rings
    • 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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  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of microorganisms, in particular to marine streptomyces and application of an anti-plant virus preparation thereof. Marine streptomycete KCB-132, the use of the marine actinomyces as an antibacterial agent; simultaneously, the microorganism can be used for further obtaining the chemical formula C20H16O2The Actetrophenone A compound prepared by the invention is derived from a fermentation product of Streptomyces marinus KCB-132, and the compound prepared by a microbial fermentation method has the characteristics of high efficiency and environmental protection.
Figure DDA0003284182080000011

Description

Actetrophenone A compound and preparation and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to an Actetrophenone A compound, and preparation and application thereof as a plant virus resistant preparation.
Background
Plant virus diseases are diseases caused by plant virus parasitism, are diseases with various types, common occurrence and serious harm in agricultural production, and are called plant cancers. Economic losses due to plant viral diseases are reported to be as high as $ 600 billion annually worldwide, with losses in food crops alone reaching as much as $ 200 billion. Since the first plant virus, Tobacco Mosaic Virus (TMV), was discovered in 1982, over 1000 plant virus species are currently in the world. Plant viruses are obligate parasites that reside in plant cells and even in the nucleus, where replication of nucleic acids (RNA or DNA) and synthesis of protein coat are carried out, constituting novel virions. Plants infected with plant viruses often exhibit symptoms of discoloration, necrosis, deformity, and the like. Plant viruses can be transmitted by vectors such as insects, mites, soil fungi, nematodes and the like, and can also be transmitted by non-vectors such as pollen, seeds and the like. The characteristics of multiple plant virus types, fast infection and propagation, wide transmission path and the like cause that the control of plant virus diseases is extremely difficult, and once the plant viruses are popular, serious loss is caused.
Disclosure of Invention
The invention aims to provide an Actetrophenone A compound, and preparation and application thereof as an anti-plant virus preparation.
In order to achieve the purpose, the invention adopts the technical scheme that:
an Actetrophenone A compound with a chemical formula of C19H16O4Having a structural formula of
Figure GDA0003597522880000011
The preparation method of the compound comprises the steps of fermenting and culturing the activated streptomyces marinus KCB-132 in an ISP2 culture medium, and purifying to obtain the compound shown in the formula I.
Further, the following steps are carried out:
1) the marine streptomycete KCB-132 is streaked and inoculated on an ISP2 solid culture medium, the culture is carried out at 28 ℃ until white spores grow out, sporophytes are taken and inoculated on an ISP2 liquid culture medium, the culture is carried out at 28 ℃, 150 and 220rpm/min, and the fermentation product is harvested after shaking culture for 10 d. Eluting the fermentation liquor by macroporous adsorption resin, and then concentrating under reduced pressure to obtain a total extract;
2) and (3) subjecting the total extract to silica gel column chromatography, performing gradient elution by using methanol/dichloromethane with the volume ratio of 0:100-100:0 as an eluent, collecting the elution component of the methanol/dichloromethane with the volume ratio of 3:97 of the eluent, subjecting the collected elution component to ODS reversed-phase column chromatography, collecting the elution part of the eluent containing 55% of organic alcohol, concentrating, and performing HPLC to prepare the compound Actetrophenone A.
The marine streptomycete KCB-132 is preserved in China general microbiological culture Collection center in 5 months and 7 days in 2020, and the preservation number is CGMCC No. 19782; this strain has been disclosed in the patent application No. 2020105708712 and is available to the public as a deposit certificate.
In the step 1), the organic solvent adopted for extraction is ethyl acetate and/or n-butanol; preferably ethyl acetate;
the eluent adopted by the macroporous adsorption resin is organic alcohol. Wherein, the organic alcohol is one of methanol and ethanol, preferably ethanol.
The eluent of the ODS reversed-phase column chromatography is organic alcohol and water; wherein, the volume ratio of the organic alcohol to the water is 0:100-100:0, and the organic agent is methanol, ethanol or acetonitrile, preferably methanol.
The macroporous adsorption resin column is low-polarity or non-polar macroporous adsorption resin, and low polarity is preferred.
The use of a compound as an anti-plant virus agent.
The plant virus is tobacco mosaic virus, tomato yellow mosaic virus, cucumber mosaic virus, etc.
The invention has the advantages that:
1. the prepared Actetrophenone A compound is derived from a fermentation product of Streptomyces marinus KCB-132, and the compound prepared by a microbial fermentation method has the characteristics of high efficiency and environmental protection;
2. the Actetrophenone A compound prepared by the invention has obvious activity of resisting plant viruses, is a novel compound which is not reported yet, has an inhibition rate of over 75 percent on mosaic viruses, can further explore an action mechanism of the compound, and is expected to be developed into a novel plant virus resisting preparation or a lead compound thereof.
Drawings
FIG. 1 is an Actetrophenone A positive ion mass spectrum (HR-ESI-MS) provided by an embodiment of the present invention.
Fig. 2 is Actetrophenone a provided in this embodiment of the present disclosure1H NMR spectrum (solvent: DMSO-d)6)。
FIG. 3 shows Actetrophenone A provided in the embodiments of the present invention13C NMR spectrum (solvent: DMSO-d)6)。
FIG. 4 shows an Actetrophenone A DEPT-135 spectrum (solvent: DMSO-d) provided in the examples of the present invention6)
FIG. 5 shows Actetrophenone A provided in the embodiments of the present invention1H-1H COSY spectrum (solvent: DMSO-d)6)。
FIG. 6 shows the spectrum of Actetrophenone A HMBC (solvent: DMSO-d)6)。
FIG. 7 shows the Actetrophenone A NOESY spectrum (solvent: DMSO-d) provided in the examples of the present invention6)。
FIG. 8 shows the Actetrophenone A HSQC spectra (solvent: DMSO-d) provided in the examples of the present invention6)。
Fig. 9 is a diffraction pattern of an Actetrophenone a X single crystal provided in the example of the present invention.
Detailed Description
The following examples are intended to illustrate the invention, but not to limit the substance of the invention.
Example 1 preparation of Actetrophenone A
1) Fermentation culture
The obtained marine streptomycete KCB-132 is streaked and inoculated on an ISP2 solid culture medium, the culture is carried out for 5 days at the temperature of 28 ℃, the inoculation amount is inoculated in an ISP2 liquid culture medium according to 10 percent of the inoculation amount, the culture is carried out at the temperature of 28 ℃ and at the speed of 150-220rpm/min, and the fermentation product is harvested after shaking culture for 10 days. Filtering the fermented product to obtain fermentation liquid and mycelium, passing the fermentation liquid through macroporous adsorbent resin column (12nm, 50 μm), eluting with 100% ethanol, and concentrating under reduced pressure (vacuum degree of 100mbar, rotation speed of 100rpm) with rotary evaporator to obtain total extract.
The ISP2 liquid culture medium comprises glucose 0.4%, yeast powder 0.4%, and malt extract powder 1%. Solid medium was supplemented with 2% agar on this basis.
2) Separating and purifying crude extract
The total extract is subjected to normal phase silica gel (200-300 mesh, 54-75 μm) column chromatography, and the content of dichloromethane: methanol is subjected to gradient elution with the flow rate of 5ml/min, and the methanol is mixed according to the volume ratio of 100:0 to 0:100 (dichloromethane: methanol (v/v)). The eluted fractions at an eluent volume ratio of 97:3 (dichloromethane: methanol) were collected, and the collected eluted fractions were subjected to ODS (C-18, 50 μm) reverse phase column chromatography with water: methanol (v/v) was gradient eluted at a flow rate of 2ml/min in a volume ratio of 100:0 to 0:100, 45: 55 (water: methanol), concentrated, and subjected to HPLC preparation with a methanol: water as mobile phase, elution rate of 1ml/min, collection retention time tRThe fraction is 15.8-17.5min to obtain the compound Actetrophenone A.
Example 2 Structure confirmation of Actetrophenone A
1. Apparatus and materials
Jasco P-1020 digital polarimeter, Agilent TOF/6500 high resolution Mass Spectrometry, Shimadzu UV-2401 visible-ultraviolet Spectrophotometer, nuclear magnetism Bruke Avance III 500NMR spectrometer.
2. Structure identification of compounds
Actetrophenone a: light brown powder, readily soluble in dimethylsulfoxide, methanol, acetone, chloroform, slightly soluble in water, uv (acetonitrile) λ max (log ∈)200(2.5),219(2.6),274(2.9),298(2.5),324(2.6),343(2.3) nm; HRESIMS [ M + H ] + at M/z 309.1122(calculated 309.1127).
The nuclear magnetic data of the Actetrophenone A are shown in the table I, and figure 1 shows an Actetrophenone A positive ion mass spectrum (HR-ESI-MS). FIG. 2 is Actetrophenone A1H NMR spectrum (solvent: DMSO-d)6). FIG. 3 is Actetrophenone A13C NMR spectrum (solvent: DMSO-d)6). FIG. 4 is an Actetrophenone A DEPT-135 spectrum (solvent: DMSO-d)6). FIG. 5 is Actetrophenone A1H-1H COSY spectrum (solvent: DMSO-d)6). FIG. 6 shows the Actetrophenone A HMBC profile (solvent: DMSO-d)6). FIG. 7 is an Actetrophenone A NOESY spectrum (solvent: DMSO-d)6). FIG. 8 shows the Actetrophenone A HSQC spectra (solvent: DMSO-d)6). FIG. 9 is a single crystal diffractogram of Actetrophenone A X.
Table 1: nuclear magnetic data for Actetrophenone A (1H NMR 500MHz, 13C NMR 125 MHz).
Figure GDA0003597522880000041
Figure GDA0003597522880000051
The structure of the compound was determined accordingly.
Example 3 preliminary evaluation of Actetrophenone A Activity against plant viruses
1) Control of tobacco mosaic virus by compound
Selecting healthy and consistent-growth common tobacco K326 with 4-5 leaf stages. The experiment was set up with a total of 5 treatments: actetrophenone A600-fold diluent, 400-fold diluent, 200-fold diluent for positive control, and clear water blank control. After 1d of application, 200-mesh quartz sand was uniformly scattered on tobacco leaf blades, and virus (100. mu.g/mL) was inoculated by mechanical rubbing. 30 tobacco seedlings are set for each treatment, and the treatment is repeated for 3 times. Disease index was calculated every 7 days after dosing.
TABLE 2 control of tobacco mosaic virus by compounds
Figure GDA0003597522880000052
Note that the positive control is 8% ningnanmycin aqua
2) Prevention and treatment effect of compound on tomato yellow leaf curl virus or cucumber mosaic virus
Common tomatoes and cucumbers are respectively selected as research objects, the prevention and control effects of the compounds on tomato yellow leaf curl virus and cucumber mosaic virus are determined according to the experimental settings, and specific results are shown in tables 3 and 4.
TABLE 3 preventive and therapeutic effects of the compounds on tomato yellow leaf curl virus
Figure GDA0003597522880000053
Figure GDA0003597522880000061
Note that the positive control is 8% ningnanmycin aqua
TABLE 4 preventive and therapeutic effects of the compounds against cucumber mosaic virus
Figure GDA0003597522880000062
Note that the positive control is 8% ningnanmycin aqua
According to the control effect data, the Actetrophenone A has better plant virus activity inhibition effect.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (5)

1. An Actetrophenone A compound characterized by: the chemical formula of the compound is C19H16O4Having a structural formula of
Figure DEST_PATH_IMAGE001
The formula I is shown.
2.A process for the preparation of a compound according to claim 1, characterized in that:
1) inoculating marine streptomycete KCB-132 on ISP2 solid culture medium by streaking, culturing at 28 ℃ until white spores grow out, inoculating sporophytes to liquid culture medium, culturing at 28 ℃, 150-; eluting the fermentation liquor by macroporous adsorption resin, and then concentrating under reduced pressure to obtain a total extract;
2) and (3) subjecting the total extract to silica gel column chromatography, performing gradient elution by using methanol/dichloromethane with the volume ratio of 0:100-100:0 as an eluent, collecting the elution component of the methanol/dichloromethane with the volume ratio of 3:97 of the eluent, subjecting the collected elution component to ODS reversed-phase column chromatography, collecting the elution part of the eluent containing 55% of organic alcohol, concentrating, and performing HPLC to prepare the compound Actetrophenone A.
3.A process for the preparation of a compound according to claim 2, characterized in that:
the eluent adopted by the macroporous adsorption resin elution in the step 1) is organic alcohol.
4. Use of a compound according to claim 1, wherein: the application of the compound as an anti-plant virus preparation.
5. Use of a compound according to claim 4, wherein: the plant virus is tobacco mosaic virus, tomato yellow mosaic virus or cucumber mosaic virus.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108821959A (en) * 2018-07-26 2018-11-16 杭州科兴生物化工有限公司 A kind of tetrahydro anthracene compound and its preparation method and application
CN111454869A (en) * 2020-06-22 2020-07-28 滨州医学院 Marine streptomyces and application thereof
CN112759569A (en) * 2019-11-06 2021-05-07 烟台蓝创生物技术有限公司 Preparation and application of Actinephthoran A and Actinephthoran B

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108821959A (en) * 2018-07-26 2018-11-16 杭州科兴生物化工有限公司 A kind of tetrahydro anthracene compound and its preparation method and application
CN112759569A (en) * 2019-11-06 2021-05-07 烟台蓝创生物技术有限公司 Preparation and application of Actinephthoran A and Actinephthoran B
CN111454869A (en) * 2020-06-22 2020-07-28 滨州医学院 Marine streptomyces and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Antibacterial and Cytotoxic Bridged and Ring Cleavage Angucyclinones From a Marine Streptomyces sp;Lin Guo等;《ORIGINAL RESEARCH》;20200804;第8卷;全文 *
Isolation, structure elucidation and racemization of (+)- and (-)-pratensilins A–C: unprecedented spiro indolinone-naphthofuran alkaloids from a marine Streptomyces sp.;Shumin Zhang等;《Chem. Commun.》;20170818;第53卷;全文 *
新型angucycline/angucyclinone类天然产物的研究进展（2010-2020）;张景琰等;《生物工程学报》;20210625;第37卷(第6期);全文 *

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