WO2023184724A1 - 一种抗霉素类化合物及其制备方法和用途 - Google Patents
一种抗霉素类化合物及其制备方法和用途 Download PDFInfo
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- compound
- antimycin
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- ethyl acetate
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- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 229930182536 Antimycin Natural products 0.000 title claims abstract description 16
- -1 Antimycin compound Chemical class 0.000 title claims abstract description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 150000001875 compounds Chemical class 0.000 claims abstract description 22
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 238000004440 column chromatography Methods 0.000 claims abstract description 10
- 241000123650 Botrytis cinerea Species 0.000 claims abstract description 8
- 241000228153 Penicillium citrinum Species 0.000 claims abstract description 8
- 239000000287 crude extract Substances 0.000 claims abstract description 8
- 241000186361 Actinobacteria <class> Species 0.000 claims abstract description 7
- 229930000044 secondary metabolite Natural products 0.000 claims abstract description 5
- 238000010828 elution Methods 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- CQIUKKVOEOPUDV-IYSWYEEDSA-N antimycin Chemical class OC1=C(C(O)=O)C(=O)C(C)=C2[C@H](C)[C@@H](C)OC=C21 CQIUKKVOEOPUDV-IYSWYEEDSA-N 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000002024 ethyl acetate extract Substances 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 238000004007 reversed phase HPLC Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 3
- 239000012141 concentrate Substances 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
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 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 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 239000013535 sea water Substances 0.000 claims description 2
- 235000002639 sodium chloride Nutrition 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 241001147825 Actinomyces sp. Species 0.000 claims 1
- 238000011081 inoculation Methods 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 238000002953 preparative HPLC Methods 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 238000010898 silica gel chromatography Methods 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000000843 anti-fungal effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 4
- 241000186046 Actinomyces Species 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- UIFFUZWRFRDZJC-UHFFFAOYSA-N Antimycin A1 Natural products CC1OC(=O)C(CCCCCC)C(OC(=O)CC(C)C)C(C)OC(=O)C1NC(=O)C1=CC=CC(NC=O)=C1O UIFFUZWRFRDZJC-UHFFFAOYSA-N 0.000 description 1
- NQWZLRAORXLWDN-UHFFFAOYSA-N Antimycin-A Natural products CCCCCCC(=O)OC1C(C)OC(=O)C(NC(=O)c2ccc(NC=O)cc2O)C(C)OC(=O)C1CCCC NQWZLRAORXLWDN-UHFFFAOYSA-N 0.000 description 1
- 239000005820 Prochloraz Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- UIFFUZWRFRDZJC-SBOOETFBSA-N antimycin A Chemical compound C[C@H]1OC(=O)[C@H](CCCCCC)[C@@H](OC(=O)CC(C)C)[C@H](C)OC(=O)[C@H]1NC(=O)C1=CC=CC(NC=O)=C1O UIFFUZWRFRDZJC-SBOOETFBSA-N 0.000 description 1
- PVEVXUMVNWSNIG-UHFFFAOYSA-N antimycin A3 Natural products CC1OC(=O)C(CCCC)C(OC(=O)CC(C)C)C(C)OC(=O)C1NC(=O)C1=CC=CC(NC=O)=C1O PVEVXUMVNWSNIG-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- ZHNUHDYFZUAESO-UHFFFAOYSA-N formamide Substances NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002678 macrocyclic compounds Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000005787 mitochondrial ATP synthesis coupled electron transport Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000001069 nematicidal effect Effects 0.000 description 1
- 239000003592 new natural product Substances 0.000 description 1
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 230000001963 piscicidal effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- TVLSRXXIMLFWEO-UHFFFAOYSA-N prochloraz Chemical compound C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl TVLSRXXIMLFWEO-UHFFFAOYSA-N 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
Classifications
-
- 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
- C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D321/00—Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
-
- 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
- A01N43/24—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 two or more hetero atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Definitions
- the present invention relates to antimycin compounds, in particular to an antimycin compound and its preparation method and use.
- Antimycin compounds are a class of natural products that share a common structural skeleton, consisting of a macrocyclic ring connected to a 3’-formamide salicylic acid unit through an amide bond. This class of compounds is usually isolated from actinomycetes and has great structural diversity based on the size of the macrocycle (9, 12, 15 or 18 membered rings) and the substituents on the ring. Since their first isolation in 1949, antimycin compounds have attracted great attention due to their powerful and diverse biological activities. In particular, due to their ability to inhibit the mitochondrial electron transport chain, antimycins have been shown to possess antifungal, insecticidal, nematicidal, and piscicidal activity. Recently, several classes of antimycin-type compounds have also been shown to possess promising anticancer and anti-inflammatory activities.
- the technical problem to be solved by the present invention is to provide an antimycin compound and its preparation method and use.
- the antimycin compound has an inhibitory effect on Botrytis cinerea and Penicillium citrinum.
- a method for preparing antimycin compounds including the following steps:
- the marine actinomycetes (Steptomyces sp.4-7) with the deposit number CCTCC NO: M 2020953 were streaked on the plate of Gao's No. 1 solid medium, cultured and activated in a 28°C incubator for 7 days, and single cells were picked.
- the colonies were inoculated into Gao's No. 1 liquid culture medium, placed on a shaker at a temperature of 28°C and a rotation speed of 180 rpm/min for bacterial enrichment. After culturing for 3 days, the seed liquid was collected, and then the seed liquid was inoculated with a volume ratio of 10%. Inoculate it into YMG medium and culture it at 28°C for 11 days to obtain the fermentation product;
- step (1) Add an equal volume of ethyl acetate to the fermentation product obtained in step (1). After repeated extraction three times, combine the ethyl acetate phases obtained by the three extractions. Concentrate the ethyl acetate extract under reduced pressure and evaporate to dryness to obtain crude extract;
- the crude extract obtained in step (2) is first dissolved in a mixed solvent of methylene chloride and methanol with a volume ratio of 1:1, then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, using a volume ratio of Use (100:1) to (0:1) petroleum ether-ethyl acetate solution as the eluent for gradient elution, collect the elution fractions, arrange them according to the polarity of the fractions from small to large, and combine to obtain 6 Component; perform gradient elution on reversed-phase medium-pressure column chromatography for the second component collected, using acetonitrile-water with a volume ratio of 30-100% as the eluent for gradient elution, and collect the elution fractions , arranged according to the polarity of the fractions from large to small, and combined to obtain 21 components; the collected 9th component was used as the mobile phase, which was a solution mixed with acetonitrile and
- the preparation method of the YMG medium described in step (1) is as follows: dissolve 20g starch, 10g glucose, 5g yeast extract, 5g malt extract, 0.5g calcium carbonate and 30g sea salt in 1000 mL sea water.
- the volume percentage of acetonitrile ranges from 30 to 100%, and the elution time is 150 minutes.
- the flow rate of the compound separation and preparation in the semi-preparative reversed-phase high-performance liquid chromatography described in step (3) is 2.0 mL/min.
- an antimycin compound and its preparation method and use are isolated from the secondary metabolites of marine actinomyces Steptomyces sp.4-7
- antimycin fermentation products are obtained through microbial fermentation culture, and then the fermentation products are soaked and extracted with ethyl acetate to obtain a crude extract, which is then passed through a medium-pressure forward silica gel column. Chromatography, medium-pressure reverse column chromatography, and reversed-phase semi-preparative high-performance liquid chromatography are separated and purified.
- the compound has significant activity against Botrytis cinerea and Penicillium citrinum, and can be used in the development of lead drugs to inhibit related plant diseases caused by Botrytis cinerea and Penicillium citrinum.
- the above-mentioned marine actinomyces (Steptomyces sp.4-7), the bacterial preservation number is CCTCC NO: M 2020953, was deposited in the China Type Culture Collection Center on December 21, 2020, and the deposit address is Wuhan University, China. .
- the preparation method of the antimycin compound shown in the structural formula (I) of Example 1 specifically includes the following steps:
- the marine actinomycetes (Steptomyces sp.4-7) with the deposit number CCTCC NO: M 2020953 were streaked on the plate of Gao's No. 1 solid medium, cultured and activated in a 28°C incubator for 7 days, and single cells were picked.
- the colonies were inoculated into Gao's No. 1 liquid culture medium, placed on a shaker at a temperature of 28°C and a rotation speed of 180 rpm/min for bacterial enrichment. After culturing for 3 days, the seed liquid was collected, and then the seed liquid was inoculated with a volume ratio of 10%. Inoculate it into YMG medium and culture it at 28°C for 11 days to obtain the fermentation product;
- the crude extract obtained in step 2 is first dissolved in a mixed solvent of methylene chloride and methanol with a volume ratio of 1:1, then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, using a volume ratio of ( Use a petroleum ether-ethyl acetate solution of 100:1) to (0:1) as the eluent for gradient elution. Collect the elution fractions, arrange them according to the polarity of the fractions from small to large, and combine to obtain 6 components.
- the collected second component is subjected to gradient elution by reversed-phase medium-pressure column chromatography, using acetonitrile-water with a volume ratio of 30-100% as the eluent for gradient elution, collecting the elution fractions, and performing gradient elution.
- Phase high performance liquid chromatography is separated and purified to obtain the compound, whose structure is shown in (I):
- Compound I of the present invention is a white powder, and high-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gives its quasi-molecular ion peak m/z 521.2480[M+H] + .
- HR-ESI-MS high-resolution mass spectrometry
- 13 C NMR spectrum the molecular formula is determined to be C 26 H 37 N 2 O 9.
- the 1 H and 13 C NMR spectrum data of this compound are shown in Table 1:
- test sample is the pure compound I isolated and purified in the above Example 1. Precisely weigh an appropriate amount of the sample and prepare a solution with the required concentration with DMSO for testing the antifungal activity.
- indicator bacteria used in this experiment were Botrytis cinerea (CGMCC 3.3789) and Penicillium citrinum (ATCC 1109).
- Oxford cup method antifungal test method Apply 100 ⁇ L of spore suspension evenly on the PDA plate, and place the Oxford cup in the center of the plate. Add 100 ⁇ L of compound solutions of different concentrations into the Oxford cup, with concentrations of 32 ⁇ g/mL, 16 ⁇ g/mL, 8 ⁇ g/mL, 4 ⁇ g/mL, 2 ⁇ g/mL, 1 ⁇ g/mL, and 0.5 ⁇ g/mL. DMSO was used as a negative control, and the same concentration of prochloraz was used as a positive control. The fungal growth was observed after culturing for 5 days at 28°C. Four culture plates were set up for each treatment, and the entire experiment was repeated three times.
- the minimum inhibitory concentration (MIC) of compound I against Botrytis cinerea (CGMCC 3.3789) was determined to be 2 ⁇ g/ml, and the MIC against Penicillium citrinum (ATCC 1109) was determined to be 4 ⁇ g/ml.
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- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
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Abstract
本发明公开了一种抗霉素类化合物及其制备方法和用途，特点是该化合物从海洋放线菌的次级代谢产物中分离得到，其结构式如Ⅰ所示，其制备方法步骤包括将保藏号为CCTCC NO:M2020953的海洋放线菌Steptomyces sp.4-7通过发酵培养来获取抗霉素化合物的发酵物，然后将发酵物用乙酸乙酯浸泡提取得粗浸膏，在此基础上将该粗浸膏经正相硅胶柱层析，反向中压柱层析和反相半制备高效液相色谱分离纯化得到，优点是该抗霉素类化合物具有抗灰葡萄孢和桔青霉的作用。
Description
本发明涉及抗霉素类化合物，尤其是涉及一种抗霉素类化合物及其制备方法和用途。
抗霉素类化合物是一类天然产物，它们拥有一个共同的结构骨架，由一个大环通过酰胺键与3’-甲酰胺水杨酸单元连接而成。这类化合物通常是从放线菌中分离出来的，基于大环(9、12、15或18元环)的大小不同以及环上的取代基不同，这些化合物具有很大的结构多样性。自1949年首次分离以来，抗霉素类化合物因其强大而多样的生物活性而受到人们的极大关注。特别地，由于其抑制线粒体电子传递链的能力，抗霉素类化合物已被证明具有抗真菌、杀虫、杀线虫和杀鱼的活性。最近，几类抗霉素型化合物也被证明具有良好的抗癌和抗炎活性。
本发明人在对海洋放线菌一株Steptomyces sp.4-7在4-7YMG培养基发酵下的乙酸乙酯提取物的化学调查中，发现了一个抗霉素类型新天然产物，将其命名为Antimycin A
7c，进一步对其次级代谢产物进行分离纯化及活性评价。目前尚未见该化合物的化学结构及活性的报道，因此市场上也尚未见有与此相关的药物。
发明内容
本发明所要解决的技术问题是提供一种抗霉素类化合物及其制备方法和用途，该抗霉素类化合物对灰葡萄孢和桔青霉具有抑制作用。
本发明解决上述技术问题所采用的技术方案为：
1、一种抗霉素类化合物，该抗霉素类化合物从海洋放线菌的次级代谢产物中分离得到，其结构式如(I)所示；
2、一种抗霉素类化合物的制备方法，包括如下步骤：
(1)发酵生产
将保藏号为CCTCC NO：M 2020953的海洋放线菌(Steptomyces sp.4-7)在高氏1号固体培养基的平板上划线，于28℃培养箱中培养活化7天后，挑取单菌落接种于高氏1号液体培养基中，于温度28℃、180rpm/min的转速下置于摇床上培养增菌，培养3天后收集种子液，然后将种子液按体积比10％的接种量接种到YMG培养基中，于温度28℃培养11天，获得发酵物；
(2)浸膏提取
在步骤(1)得到的发酵物中加入与发酵物等体积的乙酸乙酯，反复萃取3次后，合并三次萃取所得乙酸乙酯相，将乙酸乙酯提取液减压浓缩蒸干后，获得粗浸膏；
(3)化合物的分离制备
将步骤(2)得到的粗浸膏首先用体积比为1：1的二氯甲烷和甲醇混合溶剂溶解后，加200-300目硅胶拌样，进行正相中压柱层析，采用体积比为(100:1)～(0:1)的石油醚-乙酸乙酯溶液为洗脱剂进行梯度洗脱，收集洗脱流分，按流分极性由小到大排列，合并得到6个组分；将收集得到的第2个组分进行反相中压柱层析梯度洗脱，采用体积比为30-100％的乙腈-水为洗脱剂进行梯度洗脱，收集洗脱流分，按流分极性由大到小排列，合并得到21个组分；将收集得到的第9个组分采用由乙腈与水按体积比24:76的比例混合而成的溶液为流动相，经半制备反相高效液相色谱分离纯化获得化合物，其结构如(I)所示，
步骤(1)中所述的YMG培养基的配制方法如下：将20g淀粉、10g葡萄糖、5g酵母提取物、5g麦芽浸膏、0.5g碳酸钙和30g海盐溶于1000mL海水中配制而成。
步骤(3)中所述的反相中压柱层析(MPLC)梯度洗脱中乙腈体积百分数从30～100％，洗脱时间150min。
步骤(3)中所述的半制备反相高效液相色谱的化合物分离制备的流速为2.0mL/min。
3、上述抗霉素类化合物在制备灰葡萄孢和/或桔青霉抑制剂方面的用途。
与现有技术相比，本发明的优点在于：本发明一种抗霉素类化合物及其制备方法和用途，从海洋放线菌Steptomyces sp.4-7的次级代谢产物中分离出一种抗霉素类化合物，通过微生物发酵培养来获取抗霉素类的发酵物，然后通过将发酵物用乙酸乙酯浸泡提取，得粗浸膏，然后将该粗浸膏经中压正向硅胶柱层析，中压反向柱层析，及反相半制备高效液相色谱分离纯化得到。此外通过抗真菌活性的筛选，发现该化合物具有显著的抗灰葡萄孢和桔青霉活性，可用于抑制由灰葡萄孢和桔青霉引起的相关植物病害的先导药物开发方面的用途。
上述海洋放线菌(Steptomyces sp.4-7)，该菌保藏编号为CCTCC NO：M 2020953，于2020年12月21日保藏于中国典型培养物保藏中心，保藏地址为中国.武汉.武汉大学。
以下结合实施例对本发明作进一步详细描述。
实施例1
一种海洋放线菌(Steptomyces sp.4-7)发酵培养获取的抗霉素类化合物，其结构式如(I)所示：
实施例2
如实施例1结构式(I)所示的抗霉素类化合物的制备方法，具体包括如下步骤：
1、发酵生产
将保藏号为CCTCC NO：M 2020953的海洋放线菌(Steptomyces sp.4-7)在高氏1号固体培养基的平板上划线，于28℃培养箱中培养活化7天后，挑取单菌落接种于高氏1号液体培养基中，于温度28℃、180rpm/min的转速下置于摇床上培养增菌，培养3天后收集种子液，然后将种子液按体积比10％的接种量接种到YMG培养基中，于温度28℃培养11天，获得发酵物；
2、浸膏提取
在步骤1得到的发酵物中加入与发酵物等体积的乙酸乙酯，反复萃取3次后，合并三次萃取所得乙酸乙酯相，将乙酸乙酯提取液减压浓缩蒸干后，获得粗浸膏；
3、化合物的分离制备
将步骤2获得的粗浸膏首先用体积比为1：1的二氯甲烷和甲醇混合溶剂溶解后，加200-300目硅胶拌样，进行正相中压柱层析，采用体积比为(100:1)～(0:1)的石油醚-乙酸乙酯溶液为洗脱剂进行梯度洗脱，收集洗脱流分，按流分极性由小到大排列，合并得到6个组分；将收集的第2个组分进行反相中压柱层析梯度洗脱，采用体积比为30-100％的乙腈-水为洗脱剂进行梯度洗脱，收集洗脱流分，按流分极性由大到小排列，合并得到21个组分；将得到的第9个组分采用由乙腈与水按体积比24:76的比例混合而成的溶液为流动相，经半制备反相高效液相色谱分离纯化，获得化合物，其结构如(I)所示：
本发明化合物Ⅰ为白色粉末，正离子模式下的高分辨质谱(HR-ESI-MS)给出其准分子离子峰m/z 521.2480[M+H]
+。结合
13C NMR谱确定其分子式为C
26H
37N
2O
9，该化合物的
1H和
13C NMR谱数据见表1：
表1.化合物Ⅰ的1D NMR数据(DMSO-d
6)
注1：s—单重峰、d—二重峰、t—三重峰、q—四重峰、m—多重峰。
注2：
1H在600MHz NMR获得；
13C在150MHz NMR获得。
实施例2
实施例1所述抗霉素类化合物活性及应用
1、实验样品
被测样品溶液的配制：测试样品为上述实施例1中分离纯化的化合物Ⅰ纯品，精密称取适量样品，用DMSO配制成所需浓度的溶液供测试抗真菌活性。该实验使用的指示菌为灰葡萄孢(CGMCC 3.3789)和桔青霉(ATCC 1109)。
2、实验方法
牛津杯法抗真菌测试方法：将100μL的孢子悬浮液均匀涂在PDA板上，在板中央放置牛津杯。牛津杯中分别加入100μL的不同浓度的化合物溶液，浓度为32μg/mL、16μg/mL、8μg/mL、4μg/mL、2μg/mL、1μg/mL、0.5μg/mL。以DMSO为阴性对照，同时以相同浓度的咪鲜胺作阳性对照。在28℃下培养5d后观察真菌生长情况，每个处理设4个培养板，整个实验重复3次。
3、实验结果
牛津杯法抗真菌测试中，化合物Ⅰ对灰葡萄孢(CGMCC 3.3789)的最低抑制浓度(MIC)被确定为2μg/ml，对桔青霉(ATCC 1109)的MIC被确定为4μg/ml。
上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内，做出的变化、改型、添加或替换，也应属于本发明的保护范围。
Claims (6)
- 一种抗霉素类化合物，其特征在于该抗霉素类化合物从海洋放线菌的次级代谢产物中分离得到，其结构式如(I)所示；
- 一种权利要求1所述的抗霉素类化合物的制备方法，其特征在于包括如下步骤：(1)发酵生产将保藏号为CCTCC NO：M 2020953的海洋放线菌Steptomyces sp.4-7在高氏1号固体培养基的平板上划线，于28℃培养箱中培养活化7天后，挑取单菌落接种于高氏1号液体培养基中，于温度28℃、180rpm/min的转速下置于摇床上培养增菌，培养3天后收集种子液，然后将种子液按体积比10％的接种量接种到YMG培养基中，于温度28℃培养11天，获得发酵物；(2)浸膏提取在步骤(1)得到的发酵物中加入与发酵物等体积的乙酸乙酯，反复萃取3次后，合并三次萃取所得乙酸乙酯相，将乙酸乙酯提取液减压浓缩蒸干后，获得粗浸膏；(3)化合物的分离制备将步骤(2)得到的粗浸膏首先用体积比为1：1的二氯甲烷和甲醇混合溶剂溶解后，加200-300目硅胶拌样，进行正相中压柱层析，采用体积比为(100:1)～(0:1)的石油醚-乙酸乙酯溶液为洗脱剂进行梯度洗脱，收集洗脱流分，按流分极性由小到大排列，合并得到6个组分；将收集得到的第2个组分进行反相中压柱层析梯度洗脱，采用体积比为30-100％的乙腈-水为洗脱剂进行梯度洗脱，收集洗脱流分，按流分极性由大到小排列，合并得到21个组分；将收集得到的第9个组分采用由乙腈与水按体积比24:76的比例混 合而成的溶液为流动相，经半制备反相高效液相色谱分离纯化，获得化合物，其结构如(I)所示，
- 根据权利要求2所述的一种抗霉素类化合物的制备方法，其特征在于步骤(1)中所述的YMG培养基的配制方法如下：将20g淀粉、10g葡萄糖、5g酵母提取物、5g麦芽浸膏、0.5g碳酸钙和30g海盐溶于1000mL海水中配制而成。
- 根据权利要求2所述的一种抗霉素类化合物的制备方法，其特征在于步骤(3)中所述的反相中压柱层析梯度洗脱中乙腈体积从30～100％，洗脱时间150min。
- 根据权利要求2所述的一种抗霉素类化合物的制备方法，其特征在于步骤(3)中所述的半制备反相高效液相色谱的化合物分离制备的流速为2.0mL/min。
- 一种权利要求1所述的抗霉素类化合物在制备灰葡萄孢和/或桔青霉抑制剂方面的用途。
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