阅读说明：本技术 一种含二苯醚单元的18-羟基十八碳酸酯及其制备方法与应用 (18-hydroxy octadecanoic carbonate containing diphenyl ether unit and preparation method and application thereof ) 是由 熊亚红 李春远 丁唯嘉 祝钧杰 冯楠楠 于 2021-01-25 设计创作，主要内容包括：本发明公开了一种含二苯醚单元的18-羟基十八碳酸酯及其制备方法与应用。本发明含二苯醚单元的18-羟基十八碳酸酯,其分子式为C-(36)H-(54)O-9,结构式如式I所示：本发明还公开了上述化合物的制备方法和应用。本发明的二苯醚单元的18-羟基十八碳酸酯对于香蕉炭疽菌、番茄枯萎菌以及O78血清型大肠杆菌具有不同程度的抑制作用,可用作相关的植物病原菌杀菌剂或兽药制剂,而且原料可以大规模生产,应用前景广阔。(The invention discloses 18-hydroxy octadecanoic carbonate containing diphenyl ether unit and a preparation method and application thereof. The molecular formula of the 18-hydroxy octadecanoic carbonate containing diphenyl ether unit is C 36 H 54 O 9 The structural formula is shown as formula I:)

1. 18-hydroxy octadecanoic carbonate containing diphenyl ether unit is characterized in that the molecular formula is C₃₆H₅₄O₉The structural formula is shown as formula I:

2. the process for preparing 18-hydroxyoctadecanoic carbonate with diphenyl ether unit according to claim 1, characterized by comprising the steps of:

(1) culturing strain of Phoma sp L28 in strain culture medium;

(2) and (2) carrying out fermentation culture, extraction, concentration, separation and purification on the strain cultured in the step (1) in a fermentation culture medium to obtain the 18-hydroxyoctadecanoic carbonate containing diphenyl ether units.

3. The process for preparing 18-hydroxyoctadecanoic carbonate with diphenyl ether unit according to claim 2, characterized in that,

the strain culture medium in the step (1) comprises the following components in percentage by mass: 1.0-3.5% of glucose, 0.01-2.0% of yeast extract, 0.01-2.0% of peptone, 2.0-3.0% of agar, 0.2-2.5% of sodium chloride and the balance of water;

the fermentation medium in the step (2) contains the following components in percentage by mass: 0.0001-0.00025% of guaiol, 40-50% of polished round-grained rice, 0.05-1.5% of sodium chloride and the balance of water.

4. The process for preparing 18-hydroxyoctadecanoic carbonate with diphenyl ether unit according to claim 2 or 3, characterized in that,

the culture conditions of the strains in the step (1) are as follows: culturing for 5-10 days at 20-35 ℃;

the conditions of the fermentation culture in the step (2) are as follows: culturing for 25-60 days at 20-30 ℃.

5. The process for preparing 18-hydroxyoctadecanoic carbonate with diphenyl ether unit according to claim 2, characterized in that,

the organic solvent adopted in the extraction in the step (2) is one of ethyl acetate and n-butanol;

the separation and purification in the step (2) is to further purify the concentrated extract by silica gel column chromatography, gel column chromatography and High Performance Liquid Chromatography (HPLC).

6. The process for preparing 18-hydroxyoctadecanoic carbonate with diphenyl ether unit according to claim 5, characterized in that,

the silica gel column chromatography is to perform chromatographic separation in a 200-300-mesh silica gel column;

gradient elution is carried out on the silica gel column chromatography by using a mixed solvent of petroleum ether and ethyl acetate as an eluent;

the gradient elution is to gradually increase the volume percentage of the ethyl acetate from 0 to 30 percent, set 6 gradients which are respectively 0, 5 percent, 10 percent, 20 percent, 25 percent and 30 percent of eluent of petroleum ether/ethyl acetate is combined;

the gel column chromatography adopts a Sephdex LH-20 gel column, and adopts methanol as a mobile phase;

the high-efficiency liquidThe phase chromatography HPLC adopts a C18 packing chromatographic column with the particle size of 5 mu m, and the mobile phase is MeOH/H₂O; eluting with a mobile phase volume ratio and a flow rate of (v/v, 90-100: 10-0, 3.0mL/min, 0-10 min; v/v,100:0,3.0mL/min, 10-20 min), and obtaining the 18-hydroxyoctadecanoic carbonate containing diphenyl ether units in 12-14 min.

7. The process for preparing 18-hydroxyoctadecanoic carbonate with diphenyl ether unit according to claim 2, characterized in that,

when the strain culture medium in the step (1) is used, a test tube inclined plane is manufactured;

the extraction times in the step (2) are 2-5 times;

the concentration in the step (2) is reduced pressure concentration.

8. The use of 18-hydroxyoctadecanoic carbonate with diphenyl ether units according to claim 1, characterized in that the fungicide comprises phytopathogen fungicides and veterinary fungicides.

9. The use according to claim 8,

the plant pathogenic bacteria are banana anthracnose bacteria (Colletotrichum musae) and tomato Fusarium oxysporum (Fusarium oxysporum);

the veterinary bactericide is a poultry pathogenic escherichia coli bactericide;

the pathogenic Escherichia coli of the poultry is O78 serotype Escherichia coli (Escherichia coli).

10. Use according to claim 9,

the plant pathogenic bacteria bactericide is added with a solvent and/or an auxiliary agent and prepared into spray or powder;

the veterinary bactericide is prepared into a salt aqueous solution or is mixed with feed for feeding or injection so as to treat or prevent diseases caused by pathogenic escherichia coli of poultry.

Technical Field

The invention relates to the technical field of biochemical engineering, in particular to 18-hydroxyoctadecanoic carbonate containing diphenyl ether units, and a preparation method and application thereof.

Background

The microbial metabolite is one of the important sources of the antibacterial active substance, however, in recent years, the repeated discovery problem of the antibacterial active substance of the microorganism is more and more serious, and meanwhile, the drug resistance of pathogenic bacteria to the existing antibiotics is also increased continuously, so that a new method is urgently needed to be designed, and more antibacterial products with new structures are obtained as soon as possible, so that the research on new agricultural antibiotics with independent intellectual property rights is accelerated. At present, the strategy of adding exogenous chemical substances into a culture medium to induce microbial metabolism is a hot research at home and abroad, but the research on the influence effect of plant host components on the metabolism of endophytes is not related to the discovery of new antibacterial active substances, and meanwhile, the antibacterial natural products of 18-hydroxyoctadecanoic carbonates containing diphenyl ether units are rare.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide 18-hydroxyoctadecanoic carbonate containing diphenyl ether units.

Another object of the present invention is to provide a process for preparing the above-mentioned 18-hydroxyoctadecanoic carbonate containing diphenyl ether units.

The invention also aims to provide the application of the 18-hydroxyoctadecanoic carbonate containing diphenyl ether units.

The purpose of the invention is realized by the following technical scheme: 18-hydroxy octadecanoic carbonate (Phomaspether J) containing diphenyl ether unit and having molecular formula of C₃₆H₅₄O₉Knot ofThe formula is shown in formula I:

the preparation method of the 18-hydroxyoctadecanoic carbonate containing diphenyl ether unit comprises the following steps:

(1) culturing strain of Phoma sp L28 in strain culture medium;

(2) and (2) carrying out fermentation culture, extraction, concentration, separation and purification on the strain cultured in the step (1) in a fermentation culture medium to obtain the 18-hydroxyoctadecanoic carbonate containing diphenyl ether units.

Phoma sp L28 described in step (1) is disclosed in the literature "Song Huang, Jianxin Xu, Fenqi Li, Danli Zhou, Li Xu, Chunyan Li, Identification and antibacterial Activity of microorganisms from the Mangrove Fungus sp.L28, Chemistry of Natural Compounds,2017,53(2): 237-.

Preferably, the strain culture medium in the step (1) contains the following components in percentage by mass: 1.0-3.5% of glucose, 0.01-2.0% of yeast extract, 0.01-2.0% of peptone, 2.0-3.0% of agar, 0.2-2.5% of sodium chloride and the balance of water.

Preferably, the strain culture medium in step (1) is used by making a test tube slant.

Preferably, the culture conditions of the strain in step (1) are as follows: culturing for 5-10 days at 20-35 ℃.

Preferably, the fermentation medium in the step (2) contains the following components in percentage by mass: 0.0001-0.00025% of guaiol, 40-50% of polished round-grained rice, 0.05-1.5% of sodium chloride and the balance of water.

Preferably, the conditions of the fermentation culture in step (2) are as follows: culturing for 25-60 days at 20-30 ℃.

Preferably, the organic solvent used in the extraction in step (2) is one of ethyl acetate and n-butanol.

Preferably, the extraction times in the step (2) are 2-5 times.

Preferably, the concentration in step (2) is concentration under reduced pressure.

Preferably, the separation and purification in step (2) is further purifying the concentrated extract by silica gel column chromatography, gel column chromatography and High Performance Liquid Chromatography (HPLC).

Preferably, the silica gel column chromatography is performed in 200-300 mesh silica gel column.

Preferably, the silica gel column chromatography adopts a mixed solvent of petroleum ether and ethyl acetate as an eluent for gradient elution.

Preferably, the gradient elution is to gradually increase the volume percentage of the ethyl acetate from 0 to 30%, set 6 gradients of 0, 5%, 10%, 20%, 25%, 30%, respectively, and combine 30% petroleum ether/ethyl acetate eluents.

Preferably, the gel column chromatography adopts a Sephdex LH-20 gel column and adopts methanol as a mobile phase.

Preferably, the HPLC adopts a C18 packing chromatographic column with the particle size of 5 mu m, and the mobile phase is MeOH/H₂O; eluting with a mobile phase volume ratio and a flow rate of (v/v, 90-100: 10-0, 3.0mL/min, 0-10 min; v/v,100:0,3.0mL/min, 10-20 min), and obtaining the 18-hydroxyoctadecanoic carbonate containing diphenyl ether units in 12-14 min.

The application of the 18-hydroxy octadecanoic carbonate (Phomaspether J) containing diphenyl ether unit in preparing bactericide.

Preferably, the bactericide comprises a plant pathogenic bacterium bactericide and a veterinary bactericide.

Preferably, the phytopathogens are banana anthracnose (Colletotrichum musae) and tomato Fusarium oxysporum (Fusarium oxysporum).

Preferably, the veterinary bactericide is a poultry pathogenic escherichia coli bactericide.

Preferably, the pathogenic Escherichia coli of poultry is O78 serotype Escherichia coli.

Preferably, the plant pathogenic bacteria bactericide can be added with a solvent and/or an auxiliary agent to prepare spray or powder.

Preferably, the veterinary bactericide can be prepared into a salt aqueous solution or mixed with feed for feeding or injection to treat or prevent diseases caused by pathogenic escherichia coli of poultry.

Compared with the prior art, the invention has the following advantages and effects:

the 18-hydroxy octadecanoic carbonate (Phomaspether J) of diphenyl ether unit has stronger inhibiting effect on banana anthracnose (Colletotrichum musae) and tomato Fusarium oxysporum (Fusarium oxysporum), has moderate inhibiting effect on O78 serotype Escherichia coli (Escherichia coli), can be used as a related phytopathogen bactericide or veterinary drug preparation, can be produced in large scale by raw materials, and has wide application prospect.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Phoma sp L28, which is a Fungus involved in the present invention, is disclosed in the literature "Song Huang, Jianxin Xu, Fenqi Li, Danli Zhou, Li Xu, and Chunyan Li, Identification and antibacterial Activity of microorganisms from the Man grove Fungus sp.L28, Chemistry of Natural Compounds,2017,53(2): 237-.

Example 1

18-hydroxyoctadecanoic carbonate (Phomaspether J) containing diphenyl ether units was prepared by the following procedure:

(1) firstly, culturing the strain of the endophytic fungi Phoma sp L28 of the amaranthus (Myoporum bentioides A.Gray) in a strain culture medium, wherein the strain culture medium comprises 1.0 percent of glucose, 0.01 percent of yeast extract, 0.01 percent of peptone, 2.0 percent of agar, 0.2 percent of sodium chloride and the balance of water by mass percent. When in use, the strain is made into a test tube inclined plane, and the strain is cultured for 5 days at 28 ℃;

(2) then, the cultured Phoma sp L28 was subjected to fermentation culture in a fermentation medium containing 0.0001% guaiol (guaiol), 40% japonica rice, 0.05% sodium chloride, and the balance water, and allowed to stand at 20 ℃ for 60 days.

(3) Extracting the fermented strain in the step (2) with ethyl acetate for 3 times, concentrating the extract, performing chromatographic separation in a 200-300-mesh silica gel column (Qingdao ocean chemical Co., Ltd.), eluting with a petroleum ether-ethyl acetate mixed solvent, wherein the volume percentage of ethyl acetate is gradually increased from 0 to 30%, and setting 6 gradients of 0, 5%, 10%, 20%, 25% and 30% respectively; combining the eluates of 30% ethyl acetate; separating with Sephadex LH-20 gel column chromatography and methanol mobile phase; then using HPLC with 5 μm size C18 packing column as mobile phase (MeOH/H)₂O) the volume ratio and the flow rate are (v/v, 90-100: 10-0, 3.0mL/min, 0-10 min; v/v,100:0,3.0mL/min, 10-20 min), and obtaining colorless oily matter at 12-14 min, namely 18-hydroxyoctadecanoic carbonate (Phomaspether J) containing diphenyl ether units.

Example 2

18-hydroxyoctadecanoic carbonate (Phomaspether J) containing diphenyl ether units was prepared by the following procedure:

(1) firstly, culturing the strain of the Phoma amaroides (Myoporum bentioides A.Gray) endophytic fungi Phoma sp L28 in a strain culture medium, wherein the strain culture medium contains 3.0% of glucose, 1.5% of yeast extract, 1.5% of peptone, 2.5% of agar, 2.0% of sodium chloride and the balance of water. When in use, the strain is made into a test tube inclined plane, and the strain is cultured for 10 days at the temperature of 20 ℃;

(2) then carrying out fermentation culture on the cultured Phoma sp L28, wherein the used fermentation culture medium comprises 0.00025% of guaiacol, 50% of polished round-grained rice, 1.5% of sodium chloride and the balance of water, and standing and culturing for 50 days at 25 ℃;

(3) extracting the fermented strain obtained in the step (2) with n-butanol for 5 times, concentrating the extract, performing chromatographic separation in a 200-300-mesh silica gel column, and eluting with a petroleum ether-ethyl acetate mixed solvent, wherein the volume percentage of ethyl acetate is gradually increased from 0 to 30%, and 6 gradients are set, and are respectively 0, 5%, 10%, 20%, 25% and 30%; combining the 30% ethyl acetate eluate; separating with Sephadex LH-20 gel column chromatography and methanol mobile phase; then preparing high performance liquid colorSpectroscopy HPLC Using a 5 μm particle size C18-packed column, mobile phase (MeOH/H)₂O) the volume ratio and the flow rate are (v/v, 90-100: 10-0, 3.0mL/min, 0-10 min; v/v,100:0,3.0mL/min, 10-20 min), and obtaining colorless oily matter at 12-14 min, namely 18-hydroxyoctadecanoic carbonate (Phomaspether J) containing diphenyl ether units.

Example 3

18-hydroxyoctadecanoic carbonate (Phomaspether J) containing diphenyl ether units was prepared by the following procedure:

(1) firstly, culturing the strain of the Phoma amaroides (Myoporum bentioides A.Gray) endophytic fungi Phoma sp L28 in a strain culture medium, wherein the strain culture medium contains 2.0 percent of glucose, 1.0 percent of yeast extract, 1.0 percent of peptone, 2.0 percent of agar, 1.0 percent of sodium chloride and the balance of water. When in use, the strain is made into a test tube inclined plane, and the strain is cultured for 7 days at 25 ℃;

(2) performing fermentation culture on the cultured Phoma sp L28 with a fermentation culture medium of 0.00015% of guaiol (chemical component separated from the strain host plant Ixeris amara), 45% of semen oryzae Sativae, 0.08% of sodium chloride and the balance of water, and standing at 30 deg.C for 25 days;

(3) extracting the fermented strain obtained in the step (2) with n-butanol for 5 times, concentrating the extract, performing chromatographic separation in a 200-300-mesh silica gel column, and eluting with a petroleum ether-ethyl acetate mixed solvent, wherein the volume percentage of ethyl acetate is gradually increased from 0 to 30%, and 6 gradients are set, and are respectively 0, 5%, 10%, 20%, 25% and 30%; mixing 30% ethyl acetate eluate, separating with Sephadex LH-20 gel column chromatography and methanol mobile phase, and performing preparative High Performance Liquid Chromatography (HPLC) with C18 filler column with particle size of 5 μm and mobile phase volume ratio and flow rate of MeOH/H₂And (3) eluting O (v/v, 90-100: 10-0, 3.0mL/min, 0-10 min; v/v,100:0,3.0mL/min, 10-20 min), and obtaining a colorless oily substance at 12-14 min, namely the diphenyl ether unit-containing 18-hydroxyoctadecanoic carbonate (Phomaspether J).

The compound Phomaspeter J obtained in examples 1, 2 and 3 was examined for the following spectral data: HRESIMS M/z 653.3663[ M + Na ]]⁺(calcd.for C₃₆H₅₄O₉Na,653.3666)；¹H-NMR(400MHz,CD₃COCD₃)：δ11.10(s,1H),6.45(d,1.8Hz,1H),5.98(d,1.8Hz,1H),2.19(s,3H),4,45(q,7.2Hz,2H),1.39(t,7.2Hz,3H),6.59(d,3.0Hz,1H),6.57(d,3.0Hz,1H),8.65(s,1H),5.01(s,2H),3.82(s,3H),2.12(t,6.6Hz,2H),1.46(m,2H),1.25-1.31(br s,24H),1.36(m,2H),1.52(m,2H),3.56(m,6.0Hz,2H),3.46(t,1H,6.0Hz)；¹³C-NMR(100MHz,CD₃COCD₃): δ 102.9,163.3,111.8,146.6,106.8,160.1,171.2,22.1,62.3,14.6,134.6,131.8,106.9,158.8,103.7,151.6,61.8,55.9,173.2,34.5,25.6,29.8-30.5 (overlapping carbons), 26.9,33.9, 62.6.

The structural formulae of the compounds obtained in examples 1, 2 and 3 are shown below:

example 4

The compound of the invention has bacteriostatic activity tests on banana anthracnose, tomato fusarium wilt and O78 serotype escherichia coli: the Minimum Inhibitory Concentration (MIC) of Phomaspether J, the compound obtained in example 1, against banana anthracnose (Colletotrichum musae), tomato wilt (Fusarium oxysporum) and Escherichia coli serotype O78 (Escherichia coli, purchased from ATCC 35401, Yaya Biotech Co., Ltd, Shanghai, was determined by the double dilution method. And culturing the strain to be detected in a constant-temperature incubator at 28 ℃ for 24h, observing the growth condition of bacteria in the test tube, wherein the concentration corresponding to the test tube without bacteria growth is the minimum inhibitory concentration. Triazolone was used as a fungal positive control (against banana anthrax and tomato wilt) and ofloxacin was used as a bacterial positive control (against O78 serotype e. The results are shown in Table 1.

TABLE 1 antibacterial Activity of Phomaspether J (MIC, μ g/mL)

The experimental result of the embodiment 4 shows that the compound has strong growth inhibition effect on banana anthracnose bacteria and tomato fusarium wilt bacteria, and can be added with various solvents and additives to be prepared into spray or powder to be used as an agricultural bactericide; meanwhile, the compound has a moderate growth inhibition effect on O78 serotype escherichia coli, and can be prepared into various salt aqueous solutions or mixed with feed for feeding or injecting to treat or prevent diseases caused by O78 serotype escherichia coli.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.