CN115197243B - Sulfur-containing dibenzofuran alkaloid and preparation method and application thereof - Google Patents
Sulfur-containing dibenzofuran alkaloid and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 title abstract description 7
- 239000011593 sulfur Substances 0.000 title abstract description 7
- 229910052717 sulfur Inorganic materials 0.000 title abstract description 7
- 150000004826 dibenzofurans Chemical class 0.000 title description 3
- 150000001875 compounds Chemical class 0.000 claims abstract description 32
- 239000000284 extract Substances 0.000 claims abstract description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 81
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 45
- 239000012046 mixed solvent Substances 0.000 claims description 23
- 241001655175 Sorbus pohuashanensis Species 0.000 claims description 20
- 235000012072 hua qui shu Nutrition 0.000 claims description 20
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 239000003242 anti bacterial agent Substances 0.000 claims description 16
- 241000223600 Alternaria Species 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 12
- 238000010898 silica gel chromatography Methods 0.000 claims description 11
- 239000000725 suspension Substances 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 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 7
- 238000002791 soaking Methods 0.000 claims description 7
- 238000004440 column chromatography Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- 239000004495 emulsifiable concentrate Substances 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- 239000000375 suspending agent Substances 0.000 claims description 2
- 239000004562 water dispersible granule Substances 0.000 claims description 2
- 239000004563 wettable powder Substances 0.000 claims description 2
- 239000004599 antimicrobial Substances 0.000 claims 2
- 244000052616 bacterial pathogen Species 0.000 abstract description 9
- TXCDCPKCNAJMEE-UHFFFAOYSA-N dibenzofuran Chemical compound C1=CC=C2C3=CC=CC=C3OC2=C1 TXCDCPKCNAJMEE-UHFFFAOYSA-N 0.000 abstract description 8
- 241000223602 Alternaria alternata Species 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 230000012010 growth Effects 0.000 abstract description 5
- 229930013930 alkaloid Natural products 0.000 abstract description 4
- 150000003797 alkaloid derivatives Chemical class 0.000 abstract description 4
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 241001092391 Sorbus Species 0.000 abstract description 3
- 239000004480 active ingredient Substances 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 235000008345 mountainash Nutrition 0.000 abstract description 2
- 239000000575 pesticide Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 18
- 239000003814 drug Substances 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 241000196324 Embryophyta Species 0.000 description 12
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 11
- 241000208125 Nicotiana Species 0.000 description 10
- 229940079593 drug Drugs 0.000 description 10
- 239000011259 mixed solution Substances 0.000 description 10
- 241000323752 Alternaria longipes Species 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 230000000844 anti-bacterial effect Effects 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 241000588626 Acinetobacter baumannii Species 0.000 description 4
- 241000194031 Enterococcus faecium Species 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 241000191963 Staphylococcus epidermidis Species 0.000 description 4
- YZBQHRLRFGPBSL-RXMQYKEDSA-N carbapenem Chemical compound C1C=CN2C(=O)C[C@H]21 YZBQHRLRFGPBSL-RXMQYKEDSA-N 0.000 description 4
- 229940041011 carbapenems Drugs 0.000 description 4
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 241000220324 Pyrus Species 0.000 description 3
- 244000061456 Solanum tuberosum Species 0.000 description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 description 3
- -1 biphenyl phytoalexins Chemical class 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 235000021017 pears Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 229930000044 secondary metabolite Natural products 0.000 description 3
- XMAYWYJOQHXEEK-OZXSUGGESA-N (2R,4S)-ketoconazole Chemical compound C1CN(C(=O)C)CCN1C(C=C1)=CC=C1OC[C@@H]1O[C@@](CN2C=NC=C2)(C=2C(=CC(Cl)=CC=2)Cl)OC1 XMAYWYJOQHXEEK-OZXSUGGESA-N 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 2
- IHPVFYLOGNNZLA-UHFFFAOYSA-N Phytoalexin Natural products COC1=CC=CC=C1C1OC(C=C2C(OCO2)=C2OC)=C2C(=O)C1 IHPVFYLOGNNZLA-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 235000010290 biphenyl Nutrition 0.000 description 2
- 239000004305 biphenyl Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 229960003405 ciprofloxacin Drugs 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 244000000004 fungal plant pathogen Species 0.000 description 2
- 229960004125 ketoconazole Drugs 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229960003085 meticillin Drugs 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 2
- 239000000280 phytoalexin Substances 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000412366 Alternaria mali Species 0.000 description 1
- 240000005662 Aronia melanocarpa Species 0.000 description 1
- 235000007425 Aronia melanocarpa Nutrition 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 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 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 244000141359 Malus pumila Species 0.000 description 1
- 206010027146 Melanoderma Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 241001329956 Nothopassalora personata Species 0.000 description 1
- 241001676783 Preussia isomera Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000014459 Sorbus Nutrition 0.000 description 1
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 1
- 241001123668 Verticillium dahliae Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 230000001775 anti-pathogenic effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000012969 defense response to bacterium Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- OZQGLZFAWYKKLQ-UHFFFAOYSA-N oxazinane Chemical group C1CCONC1 OZQGLZFAWYKKLQ-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
- C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
- C07D513/04—Ortho-condensed systems
-
- 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
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a sulfur-containing dibenzofuran type alkaloid and a preparation method and application thereof, and belongs to the technical field of pesticide preparation. The structural formula of the compound provided by the invention is
Description
Technical Field
The invention relates to the technical field of pesticide preparation, in particular to sulfur-containing dibenzofuran type alkaloid and a preparation method and application thereof.
Background
Tobacco brown spot is a major leaf disease in the late stage of tobacco (Nicotiana tabacum) plant growth, and occurs in all tobacco producing areas of the world. According to the estimated report of the literature, the incidence area of tobacco brown spot disease in China accounts for 30% -50% of the planting area of tobacco in China each year, and the tobacco leaf disease has the largest occurrence range and the largest harm in the tobacco planting industry in China. Alternaria tabaci is caused by infection with Alternaria (Alternaria) fungi, whose major pathogenic bacteria are Alternaria tabaci (A. Alternata) and Alternaria longifolia (A. Longipes). Meanwhile, alternaria fungi is also an important plant pathogenic fungus widely distributed on the whole world, more than 40 species are about available, more than 95% of the species can be parasitic on plants, and plant diseases are caused in more than ten important crops including wheat, tobacco, potatoes, tomatoes, apples, oranges and pears, cash crops and fruit trees. The us quarantine department has found in 2003 that pears imported from our country are infected with a fungus of the genus alternaria, and thus the pears imported from our country are suspended indefinitely, resulting in a huge economic loss. Therefore, the development of the efficient, low-toxicity, natural and environment-friendly antibacterial agent has important significance for the production of cash crops and the safety of foods.
Plants often suffer from adverse factors such as physics, chemistry, biology and the like in the environment in the growth process, can cause the change of secondary metabolites of the plants and start in-vivo defense mechanisms, thereby reducing or avoiding damage to the plants, leading the plants to present certain stress resistance, and simultaneously causing the increase of the accumulation of active secondary metabolites in the plants. Such as Aronia melanocarpa (Sorbus aucu pa) infection with epidemic disease, biphenyl phytoalexins can be produced around the focus to inhibit further proliferation of erwinia fire. Recently, we have found that stimulation of Sorbus Pohuashanensis Suspension Cells (SPSCs) with yeast extract as a bioenductor induces de novo synthesis of biphenyl phytoalexins after a certain period of time. Therefore, the plant suspension cells are cultured by using a biotechnology means, and the antibacterial defense mechanism of the plant cells is started by adding the fungus extract, so that the plant cells are induced to synthesize secondary metabolites with antibacterial activity, more novel, obvious, safe and effective antibacterial substances can be found, and the method has important significance for the development of natural antibacterial agents.
Disclosure of Invention
The invention provides a sulfur-containing dibenzofuran type alkaloid, a preparation method and application thereof, and the compound provided by the invention has a novel structure and an obvious inhibition effect on Alternaria longifolia (Alternaria longipes) which is a pathogenic bacterium of tobacco brown spot.
The invention firstly provides a compound shown in a formula I or an agropharmaceutically acceptable salt thereof;
the invention also provides a preparation method of the compound shown in the formula I, which comprises the following steps:
s1: soaking and extracting dry suspension cells of Sorbus pohuashanensis with methanol or ethanol, and concentrating the extractive solution to obtain extract;
s2: performing silica gel column chromatography gradient elution on the extract obtained in the step S1 by using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent to obtain 9 components, and collecting the 4 th component;
s3: performing silica gel column chromatography gradient elution on the component collected in the step S2 by using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent, and collecting the 4 th component;
s4: and (3) carrying out Sephadex LH-20 column chromatography on the 4 th component obtained in the step (S3) by using a dichloromethane/methanol mixed solvent to obtain the compound shown in the formula I.
In the preparation method, in S2, the silica gel column chromatography gradient elution is performed by sequentially using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent with volume ratio of 200:0, 99.5:0.5, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40, 50:50;
s3, performing silica gel column chromatography gradient elution by sequentially using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent with volume ratios of 100:1, 99:1 and 98:2;
in S4, the volume ratio of the dichloromethane/methanol mixed solvent is 1:1.
In the preparation method, in S1, the soaking and extracting temperature is room temperature; the times of soaking and extracting are 1-3 times;
s3, performing gradient elution on the silica gel column for 1 time;
in S4, the number of chromatographic separations of the Sephadex LH-20 column is 2; the Sephadex LH-20 column chromatography separation further comprises a step of concentrating chromatographic liquid.
The room temperature is well known to those skilled in the art and is typically 15 to 40 ℃.
In the preparation method, the concentration is reduced pressure concentration at 40 ℃.
The application of the compound shown in the formula I or the pharmaceutically acceptable salt thereof in the preparation of the antibacterial agent also belongs to the protection scope of the invention.
Specifically, the bacteria controlled by the antibacterial agent is alternaria longifolia Alternaria longipes.
The application of the compound shown in the formula I or the pharmaceutically acceptable salt thereof in preventing and treating pathogenic bacteria infection also belongs to the protection scope of the invention.
Specifically, the pathogenic bacteria are alternaria longifolia Alternaria longipes.
The invention further provides an antibacterial agent which comprises a compound shown in the formula I or an agropharmaceutically acceptable salt thereof.
The antibacterial agent also comprises an auxiliary agent acceptable in agriculture and pharmacy.
The dosage form of the antibacterial agent is wettable powder, water dispersible granules, a suspending agent or emulsifiable concentrate.
In the antibacterial agent, the content of the compound shown in the formula I or the agropharmaceutically acceptable salt thereof is 1-99 wt%; specifically, the content of the compound shown in the formula I or the pharmaceutically acceptable salt thereof is 10-90 wt%.
The bacteria controlled by the antibacterial agent is Alternaria longifolia Alternaria longipes.
Experiments show that the compound shown in the formula I has a strong inhibition effect on the growth of Alternaria alternata (Alternaria longipes) which is a pathogenic bacterium of Alternaria alternata.
The beneficial effects of the invention are as follows:
(1) The invention provides a method for preparing sulfur-containing dibenzofuran type alkaloid-arone from a yeast-induced Sorbus pohuashanensis suspension cell, and the prepared compound has novel structure and obvious inhibition effect on tobacco brown spot pathogenic bacteria Alternaria longifolia (Alternaria longipes).
(2) The conventional method for extracting active ingredients from plant implants is generally affected by adverse factors such as long plant growth period, unstable yield and quality. The invention uses the yeast induced plant suspension cell culture technology to produce and extract the active ingredients, has the characteristics of high production speed, easily controlled growth conditions and stable yield and quality, and is beneficial to industrialized operation; and, the yield can be continuously improved by optimizing the cell culture conditions.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The preparation method of the dried Sorbus pohuashanensis suspension cells used in the following examples is the same as the preparation method of the dried Sorbus pohuashanensis suspension cells in the following documents except that Sorbus pohuashanensis is replaced with Sorbus pohuashanensis (Li Jiaxing, li Huiliang, zhou Liangyun, etc.. Yeast induces chemical components and antibacterial activity of Sorbus pohuashanensis suspension cells [ J ]. Natural product research and development, 2019,031 (012): 2071-2076.).
The silica gel column chromatography packing is 200-300 meshes.
Methicillin-resistant staphylococcus aureus (metacinllin-resistant Staphylococcus aureus), carbapenem-resistant pseudomonas aeruginosa (Carbapenems-resistant Pseudomonas aeruginosa), carbapenem acinetobacter baumannii (Carbapenems-resistant Acinetobacter baumannii), multi-drug resistant enterococcus faecium (multitug-resistant Enterococcus faecium) and multi-drug resistant staphylococcus epidermidis (multitug-resistant Staphylococcus epidermidis) are described in the literature ((±) -prenomide: A new alkaloid featuring a rare naturally occurring tetrahydro-2H-1,2-oxazin skeleton from an endophytic fungus Preussia isomera by using OSMAC strategy, hai-Li Chen et al, fitotepia, 141 (2020) 104475), and are available to the public from the university of army of medicine, or from the institute of chinese traditional chinese medicine, the applicant, via the university of army of medicine, and are used only for relevant experiments in duplicate and not as other uses.
EXAMPLE 1 preparation of Sorbus pohuashanensis A
1. Preparation method
S1: soaking and extracting dried suspension cells of Sorbus pohuashanensis with methanol at room temperature for 3 times, mixing extractive solutions, and concentrating under reduced pressure at 40deg.C to obtain extract 6kg;
s2: sequentially performing silica gel column chromatography gradient elution on the extractum obtained in the step S1 by using dichloromethane/methanol mixed solvents with volume ratios of 200:0, 99.5:0.5, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40 and 50:50 to obtain 9 components, and collecting the 4 th component;
s3: sequentially performing silica gel column chromatography gradient elution on the components collected in the step S2 by using a dichloromethane/methanol mixed solvent with the volume ratio of 100:1, 99:1 and 98:2, and collecting the 4 th component in the components for 1 time;
s4: separating the 4 th component obtained by S3 treatment by Sephadex LH-20 column chromatography with dichloromethane/methanol mixed solvent at volume ratio of 1:1 for 2 times, and concentrating the chromatographic liquid at 40deg.C under reduced pressure to obtain 5mg of Sorbus pohuashanensis extract.
2. Analysis of results
And carrying out mass spectrum, ultraviolet and infrared analysis on the obtained white powder finished product of the arone A, wherein the analysis results are as follows:
sorbus pohuashanensis extract formula C 14 H 9 NO 3 S；ESIMS(pos.)：m/z 294[M+Na] + ，565[2M+Na] + ；HRESIMS(neg.)：m/z 270.0231[M-1] - (270.0230calcd for C 14 H 8 NO 3 S)；UV(MeOH)λ max ：230(sh)，305，330(sh)nm；IR(KBr)ν max ：3235，2956，2922，2852，1735，1623，1587，1494，1454，1385，1313，1251，1212，1029，969，939cm -1 .
Nuclear magnetic resonance analysis was performed on Sorbus pohuashanensis A, and the results are shown in Table 1.
TABLE 1 Nuclear magnetic resonance Spectroscopy data for Sorbus pohuashanensis A (δin ppm, J in Hz)
By combining the mass spectrum, ultraviolet, infrared and nuclear magnetic resonance analysis results, the prepared arone A has the following structure:
example 2
1. In order to examine the anti-pathogenic fungus effect of the prepared compound, five pathogenic fungi including potato verticillium wilt (Verticillium dahliae) ATCC 42216, alternaria mali ATCC 6663, gibberella wheat (Gibberella saubinetii) ATCC 20193, alternaria longifolia (Alternaria longipes) ATCC 26293 and black spot cabbage (Cercospora personata) ATCC 18592 are selected, and the antibacterial activity of the Sorbus pohuashanensis A is determined by a double dilution method.
Compound solution preparation: dissolving the compound prepared in example 1, namely the arone and the positive control ketoconazole in DMSO to prepare a solution with the concentration of 10 mg/mL;
preparing PDB culture solution: 25g of PDB (the ingredients are 5.0g/L of potato extract powder, 20.0g/L of glucose and 0.1g/L of chloramphenicol, shanghai Bo microorganism science and technology Co., ltd.) is taken, 1000mL of distilled water is added, heated, boiled and dissolved, and sterilized at 121 ℃ for 20 minutes for standby;
preparing a test bacterial liquid: inoculating plant pathogenic fungi stored in a 4 ℃ inclined plane into PDB culture solution, and culturing for 3-4d at 28 ℃ and 160 r/min;
the experimental method comprises the following steps: the test bacterial liquid is diluted 100 times by PDB culture liquid. In a 96-well plate, 198. Mu.L of the test bacterial liquid after dilution is added to each well of the first row. Adding 100 mu L of diluted test bacterial liquid into each row of holes; adding 2 mu L of compound solution in the first row, sucking 100 mu L of mixed solution to the second row after the mixed solution is uniformly sucked and mixed by a pipette, sucking 100 mu L of mixed solution to the third row after the mixed solution is uniformly mixed, and discarding the sucked mixed solution in the last row after the last rows are operated as above; the concentration of each row of compounds is 0.1mg/mL,0.05mg/mL,0.025mg/mL and 0.0125mg/mL, which are gradually decreased. One positive control and negative control per plate (2 μl ketoconazole solution and 2 μl DMSO solution, respectively).
And (3) judging results: culturing at 28 deg.C for 72 hr, and observing. The minimum concentration of compound without fungal growth was the Minimum Inhibitory Concentration (MIC) and the experimental results are shown in table 2.
TABLE 2 antifungal Activity of Sorbus pohuashanensis A (MIC, μg/mL)
As can be seen from Table 2, the arone has obvious inhibitory activity on Alternaria alternata (Alternaria longipes) which is a pathogenic bacterium of Alternaria alternata.
2. In order to examine the effect of the prepared compound on drug-resistant pathogenic bacteria, 5 drug-resistant bacteria (provided by army medical university) of methicillin-resistant staphylococcus aureus (Methicin-resistant Staphylococcus aureus), carbapenem pseudomonas aeruginosa (Carbapenems-resistant Pseudomonas aeruginosa), carbapenem acinetobacter baumannii (Carbapenems-resistant Acinetobacter baumannii), multi-drug-resistant enterococcus faecium (multi drug-resistant Enterococcus faecium) and multi-drug-resistant staphylococcus epidermidis (multi drug-resistant Staphylococcus epidermidis) are selected, and the antibacterial activity of the compound of the mountain ash methyl is measured by a double dilution method.
Compound solution preparation: the compound of Sorbus pohuashanensis A prepared in example 1 and positive control ciprofloxacin were dissolved in DMSO to prepare a 10mg/mL solution;
preparing LB culture solution: taking LB 25g (with the components of 10.0g/L tryptone, 5.0g/L yeast powder and 10.0g/L sodium chloride, shanghai Bo microorganism science and technology Co., ltd.), adding 1000mL of distilled water, heating, boiling for dissolution, and sterilizing at 121 ℃ for 15 minutes for later use;
preparing a test bacterial liquid: the drug-resistant bacteria are inoculated into LB culture solution from a freezing tube, and the culture solution is cultivated for 10 hours at a constant temperature of 37 ℃ and 160r/min until the culture solution becomes turbid.
The experimental method comprises the following steps: diluting the test bacterial liquid by 1000 times with LB culture liquid; in a 96-well plate, 198. Mu.L of the test bacterial liquid after dilution is added to each well of the first row. Adding 100 mu L of diluted test bacterial liquid into each row of holes; adding 2 mu L of compound solution in the first row, sucking 100 mu L of mixed solution to the second row after the mixed solution is uniformly sucked and mixed by a pipette, sucking 100 mu L of mixed solution to the third row after the mixed solution is uniformly mixed, and discarding the sucked mixed solution in the last row after the last rows are operated as above; the concentration of each row of compounds is 0.1mg/mL,0.05mg/mL,0.025mg/mL and 0.0125mg/mL, which are gradually decreased. One positive control and negative control per plate (2 μl ciprofloxacin solution and 2 μl DMSO solution, respectively).
And (3) judging results: culturing at 37 deg.C for 12 hr, and observing. The minimum compound concentration at which no bacteria grow is found to be the Minimum Inhibitory Concentration (MIC), and the experimental results are shown in table 3.
TABLE 3 antibacterial Activity of Sorbus pohuashanensis A (MIC, μg/mL)
As can be seen from Table 3, the Sorbus pohuashanensis A has no obvious inhibitory activity on 5 kinds of drug-resistant bacteria.
In conclusion, the results of this example fully demonstrate that the sulfur-containing dibenzofuran alkaloid, i.e., arone, has potential application value in further development into antifungal agents.
Claims (9)
1. A compound of formula i:
formula I.
2. A process for the preparation of a compound of formula i according to claim 1, comprising the steps of:
s1: soaking dried yeast-induced Sorbus pohuashanensis suspension cells in methanol or ethanol, and concentrating the extractive solution to obtain extract;
s2: performing silica gel column chromatography gradient elution on the extract obtained in the step S1 by using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent to obtain 9 components, and collecting the 4 th component;
s3: performing silica gel column chromatography gradient elution on the component collected in the step S2 by using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent, and collecting the 4 th component;
s4: and (3) carrying out Sephadex LH-20 column chromatography on the 4 th component obtained in the step (S3) by using a dichloromethane/methanol mixed solvent to obtain the compound shown in the formula I.
3. The preparation method according to claim 2, characterized in that: s2, performing silica gel column chromatography gradient elution by sequentially using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent with volume ratios of 200:0, 99.5:0.5, 99:1, 98:2, 90:10, 80:20, 70:30, 60:40 and 50:50;
s3, performing silica gel column chromatography gradient elution by sequentially using a dichloromethane/methanol mixed solvent or a chloroform/methanol mixed solvent with volume ratios of 100:1, 99:1 and 98:2;
in S4, the volume ratio of the dichloromethane/methanol mixed solvent is 1:1.
4. A method of preparation according to claim 2 or 3, characterized in that: s1, soaking and extracting at room temperature; the times of soaking and extracting are 1-3 times;
s3, performing gradient elution on the silica gel column for 1 time;
in S4, the number of chromatographic separations of the Sephadex LH-20 column is 2; the Sephadex LH-20 column chromatography separation further comprises a step of concentrating chromatographic liquid.
5. Use of a compound of formula i according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of an antibacterial agent;
the bacteria controlled by the antibacterial agent are Alternaria longifolia @Alternaria longipes）。
6. An antimicrobial agent characterized by: comprising a compound of formula I as defined in claim 1 or an agropharmaceutically acceptable salt thereof.
7. An antimicrobial agent according to claim 6, wherein: the dosage form of the antibacterial agent is wettable powder, water dispersible granules, suspending agent or emulsifiable concentrate.
8. The antibacterial agent according to claim 6 or 7, characterized in that: the antibacterial agent contains 1-99 wt% of the compound shown in the formula I in claim 1 or the agropharmaceutically acceptable salt thereof.
9. The antibacterial agent according to claim 6 or 7, characterized in that: the bacteria controlled by the antibacterial agent are Alternaria longifolia @Alternaria longipes）。
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