Fatty acid compound as well as preparation method and application thereof

Abstract

The invention relates to a fatty acid compound as well as a preparation method and application thereof, and the fatty acid compound has a structure as shown in a formula (I), the fatty acid compound is applied to prevention and treatment of plant fungal diseases, the fatty acid compound can inhibit the growth activity of fusarium graminearum, colletotrichum gloeosporioides, alternaria alternata and peanut rhododendron versicolor, the activity of the fatty acid compound on the alternaria alternata and the peanut rhododendron versicolor is superior to that of a positive drug carbendazim, and the fatty acid compound can be applied to prevention and treatment of plant fungal diseases. A novel compound entity is provided for effective prevention and treatment of plant fungal diseases.

Description

Technical Field

[0001] The present invention relates to a fatty acid compound and a preparation method and application thereof. Background Art

[0002] Natural products, with their unique chemical structures and significant biological activities, have become an important resource for the development of innovative drugs and agricultural biopharmaceuticals. As a key source of active natural products, fungi produce secondary metabolites characterized by high yields, ease of industrial-scale preparation, diverse chemical structures, and high biological activity. Many fungal secondary metabolites possess outstanding antibacterial, antifungal, antiviral, herbicidal, and anti-inflammatory activities, possessing high drug development potential and research value, and have become a hot topic in the current development of natural medicines.

[0003] In agricultural production, chemical synthetic pesticides have powerful pest and disease control capabilities, greatly reducing crop losses. However, due to their high dosage, long degradation cycle, and lack of precise targeting of pests and diseases, chemical synthetic pesticides have caused serious damage to soil microbial diversity, affecting soil nutrient cycles and disrupting ecological balance. At the same time, excessive residues of chemical synthetic pesticides pose a serious threat to food safety. Natural products, especially fungal secondary metabolites, with their unique structures, environmental friendliness, and diverse biological activities, provide new templates and ideas for the structural optimization and innovation of pesticide molecules, showing great development and application potential in the field of pesticide molecule design.

[0004] CN104277982B discloses a method of producing a fungus from the genus Penicillium Penicillium bilaiae Tricyclic sesquiterpenoids prepared by fermentation (CGMCC No. 9542) have inhibitory activity against the agricultural fungus Colletotrichum citrifolia and can be used as inhibitors or fungicides.

[0005] CN103243029A discloses a biocontrol fungus Trichoderma viride ( Trichoderma virens )SS161 (CGMCC accession number: 7224). This strain's fermentation product contains antifungal substances that exhibit strong antagonistic effects against a variety of pathogenic fungi. The isolation of this strain provides an excellent starting point for the research and development of microbial pesticides. Summary of the Invention

[0006] The present invention aims at providing a fatty acid compound and a preparation method and application thereof; the fatty acid compound of the present invention can prevent and control plant fungal diseases.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a fatty acid compound having a structure shown in formula (I); (I).

[0008] A second aspect of the present invention provides a method for preparing the fatty acid compound of the present invention, the method comprising: (1) Aspergillus japonicus Aspergillus japonicus The TE-739D strain was inoculated into a potato glucose liquid medium for cultivation to obtain a fermentation broth; (2) The fermentation broth is extracted with ethyl acetate, and the extract is concentrated to obtain a crude extract; (3) The crude extract is dissolved in an alcohol solvent, and the solution is mixed with silica gel and then subjected to gradient elution by vacuum column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate, and the fractions eluted by the eluent with a volume ratio of petroleum ether to ethyl acetate of 50:50 are collected; (4) The above components were subjected to reverse silica gel column chromatography gradient elution, and the elution system was methanol-water. The components eluted by the elution system with a methanol volume ratio of 60% were collected and purified to obtain the fatty acid compounds.

[0009] The third aspect of the present invention provides a use of the fatty acid compound of the present invention in the prevention and treatment of plant pathogenic fungi.

[0010] Through the above technical solution, (1) the present invention provides a Japanese Aspergillus Aspergillus Japanese New structural fatty acid compounds derived from TE-739D; (2) The present invention provides a method for preparing the novel fatty acid compound; the preparation method provided by the present invention can efficiently separate and prepare the fatty acid compound; (3) The present invention provides an application of the fatty acid compound for the prevention and treatment of plant fungal diseases. The fatty acid compound can inhibit the growth activity of wheat ergot, cucumber anthracnose, tobacco brown spot pathogen and peanut white spot pathogen, and its activity against tobacco brown spot pathogen and peanut white spot pathogen is better than that of the positive drug carbendazim, providing a new compound entity for the effective prevention and treatment of plant fungal diseases.

[0011] Biological Deposits The Aspergillus japonicus of the present invention ( Aspergillus japonicus TE-739D strain is deposited by China General Microbiology Center under the China Culture Collection Administration under the deposit number CGMCC No. 40901 and is classified as Aspergillus japonicus. Aspergillus japonicus Deposit date: October 25, 2023, deposit location: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1This is a high-resolution mass spectrum of the fatty acid compound japonione C described in the present invention.

[0013] Figure 2 The hydrogen spectrum (600 MHz, DMSO- d 6) Figure.

[0014] Figure 3 The carbon spectrum (150 MHz, DMSO- d 6) Figure. DETAILED DESCRIPTION

[0015] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0016] The first aspect of the present invention provides a fatty acid compound having a structure shown in formula (I); (I).

[0017] A second aspect of the present invention provides a method for preparing the fatty acid compound, the method comprising: (1) Aspergillus japonicus Aspergillus japonicus The TE-739D strain was inoculated into a potato glucose liquid medium for cultivation to obtain a fermentation broth; (2) The fermentation broth is extracted with ethyl acetate, and the extract is concentrated to obtain a crude extract; (3) The crude extract is dissolved in an alcohol solvent, and the solution is mixed with silica gel and then subjected to gradient elution by vacuum column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate, and the fractions eluted by the eluent with a volume ratio of petroleum ether to ethyl acetate of 50:50 are collected; (4) The above components were subjected to reverse silica gel column chromatography gradient elution, and the elution system was methanol-water. The components eluted by the elution system with a methanol volume ratio of 60% were collected and purified to obtain the fatty acid compounds.

[0018] The culture conditions include: a temperature of 25-30° C. and a culture time of 20-40 days. In the embodiment of the present invention, culture at 28° C. for 30 days is used as an example.

[0019] According to a preferred embodiment of the present invention, in step (2), the extraction is carried out under ultrasonic conditions, preferably, the ultrasonic power is 400-1200 W, and the ultrasonic time is 10 min-120 min.

[0020] According to a preferred embodiment of the present invention, in step (3), the alcohol solvent is selected from methanol and / or ethanol.

[0021] In the present invention, there is no particular limitation on the silica gel. According to a preferred embodiment of the present invention, 100-200 mesh silica gel is used for mixing the sample.

[0022] According to a preferred embodiment of the present invention, in step (3), gradient elution is performed using a petroleum ether-ethyl acetate mixture with a volume ratio of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100, and the fraction eluted with the petroleum ether-ethyl acetate mixture with a volume ratio of 50:50 is collected.

[0023] According to a preferred embodiment of the present invention, in step (4), gradient elution is performed by Lobar LiChroprep RP-18 reverse silica gel column chromatography, and a methanol-water elution system with a methanol volume ratio of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% is used for the gradient elution, and the components eluted by the methanol-water elution system with a methanol volume ratio of 60% are collected.

[0024] According to a preferred embodiment of the present invention, the purification is carried out using a semi-preparative high performance liquid chromatography, and preferably, the purification elution system is 35% by volume acetonitrile-water.

[0025] The third aspect of the present invention provides a use of the fatty acid compound in the prevention and treatment of plant pathogenic fungi.

[0026] According to a preferred embodiment of the present invention, the fungus is selected from the group consisting of Sclerotium rolfsii Sacc, Gibberella zeae Fusarium gramineae schw, cucumber anthracnose Colletotrichum to circle Arx, tobacco brown spot pathogen Alternaria alternata (Fries) Keisslar one or more; the fatty acid compound of the present invention can inhibit the growth activity of wheat fusarium, cucumber anthracnose fungus, tobacco brown spot fungus and peanut white fungus, and its activity against tobacco brown spot fungus and peanut white fungus is better than that of the positive drug carbendazim, providing a new compound entity for the effective prevention and control of plant fungal diseases.

[0027] According to a preferred embodiment of the present invention, in the application of fatty acid compounds in resisting plant pathogenic fungi, the concentration of the fatty acid compounds is 0.1-128 μg / mL, for example, it can be 0.125 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 0.8 μg / mL, 1 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, or 125 μg / mL.

[0028] The present invention will be described in detail below through examples.

[0029] The specific process of preparing the fatty acid compound of the present invention is: (1) In a sterile operating table, pick up the activated Aspergillus japonicus Aspergillus japonicus TE-739D mycelium was inoculated onto PDA plates and cultured in a constant temperature incubator at 28°C for 5 days. After incubation, a 1 cm × 1 cm colony was excised from the PDA plates and transferred to 1 L Erlenmeyer flasks (300 mL per flask) containing autoclaved potato dextrose water (PotatoDextrose Water; Brand: Haibo Biotechnology; No.: HB0233-4). (2) The inoculated Erlenmeyer flask was placed under normal temperature for 30 days for fermentation, with 12 h / day of light and 12 h / day of darkness. After fermentation, the culture medium was ultrasonically extracted with ethyl acetate (ultrasonic power 800 W, ultrasonic working time 30 min), and concentrated under vacuum (vacuum degree 0.09 MPa) to obtain a crude extract. (3) Dissolve the crude extract in methanol and add 100-200 mesh silica gel to mix the sample (mass ratio of extract to silica gel is 1:1). Then separate the components by silica gel column chromatography under reduced pressure (vacuum degree 0.05 MPa) using petroleum ether-ethyl acetate mixtures with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100. (4) The components eluted with a 50:50 mixture of petroleum ether and ethyl acetate were subjected to gradient elution on a Lobar LiChroprep RP-18 reversed-phase silica gel column using a methanol-water mixture (methanol volume ratios of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, respectively).

[0030] (5) The fraction eluted with a 60% methanol-40% water mixture was purified using a semi-preparative high performance liquid chromatography (volume ratio, 35% acetonitrile-75% water), and the fatty acid compound was finally obtained, named japonione C, with the structural formula shown in Formula (I).

[0031] The molecular structure of the fatty acid compound japonione C was determined by high-resolution mass spectrometry and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy. The physicochemical properties of the fatty acid compound japonione C are as follows: Properties: colorless oil; Solubility: easily soluble in DMSO and methanol; Molecular formula: C 12 H 20 O3; UV absorption spectrum λ max 238nm; High resolution mass spectrometry (HRESIMS): m / z 213.1492 [M + H] + (Theoretical value C 12 H 20 O3 + , 213.1485); the H NMR and C NMR data are shown in Table 1.

[0032] Table 1. NMR data of the fatty acid compound japonione C (DMSO- d 6)

[0033] Among them, the corresponding C number is as shown in formula (II), (II).

[0034] Figure 1 This is a high-resolution mass spectrum of the fatty acid compound japonione C described in the present invention.

[0035] Figure 2 The hydrogen spectrum (600 MHz, DMSO- d 6) Figure.

[0036] Figure 3 The carbon spectrum (150 MHz, DMSO- d 6) Figure.

[0037] The growth rate method was used to evaluate the inhibitory effect of the novel fatty acid compound japonione C on four plant pathogenic fungi. The specific process was as follows: The activity against plant pathogenic fungi was determined by the minimum inhibitory concentration (MIC) method, which is the lowest drug concentration that can inhibit the growth of pathogenic fungi in vitro.

[0038] The four plant pathogenic fungi tested were Peanut sphaeroides Sclerotium rolfsii Sacc, Gibberella zeae Fusarium gramineae schw, cucumber anthracnose Colletotrichum orbiculare Arx, tobacco brown spot pathogen Alternaria alternata (Fries) Keisslar. The above plant pathogenic fungi were provided by the College of Chemistry and Pharmacy, Qingdao Agricultural University.

[0039] The above plant pathogenic fungi were inoculated into PDB medium and cultured in a constant temperature incubator (28°C, 200 rpm) for 4 days. 6 CFU / mL for MIC determination. Dissolve approximately 1 mg of the compound japonione C in approximately 100 μL of DMSO, mix thoroughly, and dilute to a final concentration of 2560 μg / mL. This is used as the sample solution. Carbendazim is used as a positive control. Add 10 μL of japonione C (or the positive control carbendazim) and 190 μL of bacterial solution to the first well of a 96-well microplate. Add 100 μL of bacterial solution to the remaining 10 wells. Pipette well one well and transfer 100 μL to the second well. Reduce the sample concentration by half sequentially. The final sample concentrations in each well are 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, respectively. In a 96-well microplate, different concentrations of the compound are added to a suspension of the test bacteria. After incubation, the compound is observed. If the indicator bacteria grow in a certain well, the drug concentration in that well is ineffective in inhibiting its growth. The liquid in that well becomes turbid, and the transmittance decreases significantly. Conversely, the liquid in that well becomes clear, and the transmittance decreases insignificantly. The lowest sample concentration that completely inhibits the growth of the indicator bacteria in a small well is the compound's MIC (Microbial Inhibitor). The results are shown in Table 2.

[0040] Table 2. Growth inhibitory activity of the fatty acid compound japonione C against four plant pathogenic fungi (MIC, μg / mL)

[0041] The fatty acid compound japonione C exhibits strong inhibitory activity against both peanut white spot pathogen and tobacco brown spot pathogen. Japonione C exhibits superior activity against tobacco brown spot pathogen (MIC = 4 μg / mL) compared to the positive inhibitor carbendazim (MIC = 16 μg / mL); and superior activity against peanut white spot pathogen (MIC = 4 μg / mL) compared to the positive inhibitor carbendazim (MIC = 16 μg / mL).

[0042] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A fatty acid compound, characterized in that Having the structure shown in formula (I); （I）。 2. The method for preparing the fatty acid compound according to claim 1, wherein The method includes: (1) Aspergillus japonicus ( Aspergillus japonicus ) TE-739D strain, inoculated into potato glucose liquid medium for cultivation to obtain a fermentation broth; the Aspergillus japonicus ( Aspergillus japonicus TE-739D strain is deposited with the China General Microbiology Center under the China Culture Collection Administration, with the deposit number CGMCC No. 40901. (2) The fermentation broth is extracted with ethyl acetate, and the extract is concentrated to obtain a crude extract; (3) The crude extract is dissolved in an alcohol solvent, and the solution is mixed with silica gel and then subjected to gradient elution by vacuum column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate, and the fractions eluted by the eluent with a volume ratio of petroleum ether to ethyl acetate of 50:50 are collected; (4) The above components were subjected to reverse silica gel column chromatography gradient elution, and the elution system was methanol-water. The components eluted by the elution system with a methanol volume ratio of 60% were collected and purified to obtain the fatty acid compounds.

3. The preparation method according to claim 2, wherein The culture conditions include: a temperature of 25-30° C. and a culture time of 20-40 days.

4. The preparation method according to claim 2, wherein In step (2), the extraction is carried out under ultrasonic conditions. Preferably, the ultrasonic power is 400-1200 W and the ultrasonic time is 10 min-120 min.

5. The preparation method according to claim 2, wherein In step (3), the alcohol solvent is selected from methanol and / or ethanol; and / or Use 100-200 mesh silica gel to mix the sample; and / or Gradient elution was performed using a petroleum ether-ethyl acetate mixture with a volume ratio of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100, and the fractions eluted with a petroleum ether-ethyl acetate mixture with a volume ratio of 50:50 were collected.

6. The preparation method according to claim 2, wherein In step (4), gradient elution is performed by Lobar LiChroprep RP-18 reverse silica gel column chromatography using a methanol-water elution system with a methanol volume ratio of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, respectively, and the components eluted by the methanol-water elution system with a methanol volume ratio of 60% are collected.

7. The preparation method according to claim 2, wherein The purification is carried out using a semi-preparative high performance liquid chromatography, and preferably, the purification elution system is 35% by volume acetonitrile-water.

8. Use of the fatty acid compound according to claim 1 in controlling plant pathogenic fungi.

9. The use according to claim 8, wherein: The fungus is selected from the group consisting of Sclerotium rolfsii Sacc, Gibberella zeae Fusarium graminearum schw, cucumber anthracnose Colletotrichum orbiculare Arx, tobacco brown spot pathogen Alternaria alternate (Fries) One or more of Keisslar.

10. The use according to claim 8 or 9, wherein: The concentration of fatty acid compounds was 0.1-128 μg / mL.

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