Polyketone compound japondiene D as well as preparation method and application thereof

Abstract

The invention discloses a polyketone compound japondiene D as well as a preparation method and application thereof, and belongs to the technical field of microbial metabolites. According to the present invention, the Aspergillus japonicus TE-739D is found, the polyketone compound japondiene D is successfully separated, extracted and prepared from the metabolic fermentation product of the Aspergillus japonicus TE-739D, and through the SciFindern database retrieval and compared with the currently reported compound, the polyketone compound japondiene D is the new structure compound reported for the first time, and has the following advantages that: the new structure is the new structure compound reported for the first time; functional studies find that the polyketone compound japondiene D has a strong inhibition effect on germination of amaranthus retroflexus seeds, the effect is better than that of a positive drug glyphosate, and technical support is provided for further development of biopesticides.

Description

Technical Field

[0001] This invention relates to the field of microbial metabolite technology, and more specifically to a polyketide compound japonidiene D, its preparation method, and its applications. Background Technology

[0002] Microbial metabolites are chemical substances produced by microorganisms (such as bacteria, fungi, and actinomycetes) through their metabolic activities during growth, reproduction, or response to environmental changes. These products are not only crucial for microbial survival and adaptation but also widely used in medicine, agriculture, industry, and food. Among these, fungal metabolites account for about half of all microbial metabolites and possess advantages such as high yield, novel structure, significant activity, high "innovation index," and high "drug-likeness," making them a new highlight in current research on microbial natural product pesticides. In-depth exploration of novel agricultural lead compounds in fungi will become a breakthrough in the creation and development of novel microbial natural product pesticides.

[0003] Polyketides are naturally occurring secondary metabolites produced by bacteria, fungi, plants, and animals. In terms of bioactivity, studies have found that some polyketides significantly inhibit weed growth, effectively controlling weed growth and even causing weed death. Naturally derived polyketides have significant advantages and potential as biopesticides in weed control. Compared to chemically synthesized herbicides, they are environmentally friendly, have less impact on ecological balance, and reduce the risk of environmental pollution. Furthermore, polyketides possess rich structural diversity; through structural modification and optimization, it is expected to obtain bioherbicides with higher activity and greater selectivity, showing broad application prospects.

[0004] However, at present, there are relatively few types of polyketide compounds from natural sources with herbicidal functions, resulting in poor selectivity.

[0005] Therefore, how to enrich the variety of polyketide compounds with herbicidal functions and improve the selectivity of microbial natural product pesticides is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention has successfully extracted and prepared a polyketide compound, japonidiene D, derived from Aspergillus japonicus TE-739D, and found that it has a superior inhibitory effect on the germination of roots and shoots of Amaranthus retroflexus seeds, providing technical support for the further development of biopesticides.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A polyketide compound, japonidiene D, has the molecular formula C0. 22 H 28 O6, the structural formula is as follows:

[0009]

[0010] Another object of the present invention is to provide: the application of Aspergillus japonicus TE-739D in the preparation of the polyketide compound japonicide D, wherein Aspergillus japonicus TE-739D is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCCNO.40901.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned polyketide compound japonicine D, comprising the following steps:

[0012] (1) Expanded culture: The mycelium of Aspergillus japonicus TE-739D was inoculated into PDA plate medium and placed at 28℃ for 5 days for expanded culture;

[0013] (2) Fermentation culture: Cut off the bacterial blocks from the PDA plate medium in step (1), transfer them to potato glucose water medium, and place them under the conditions of room temperature and light / dark for 12h / 12h, and let them ferment for 30-35 days.

[0014] (3) Preparation of crude extract: The culture medium after fermentation in step (2) was extracted with ethyl acetate by ultrasonic extraction and concentrated under vacuum to prepare crude extract.

[0015] (4) Initial separation of components: After dissolving the crude extract from step (3) in methanol, silica gel was added and mixed. The components were then separated by vacuum silica gel column chromatography. The mixtures were eluted with 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, and methanol-dichloromethane mixtures with volume ratios of 10:90, 20:80 and 30:70. Similar components were combined by thin-layer chromatography to obtain seven components Fr.1-Fr.7 with increasing polarity.

[0016] (5) Component reseparation: The component Fr.4 eluted in step (4) by a mixture of petroleum ether and ethyl acetate at a volume ratio of 50:50 was eluted by reverse silica gel column chromatography gradient elution. The elution system was methanol-water with volume ratios of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. The eluted components were detected by thin-layer chromatography, and similar components were combined to obtain 6 sub-components Fr.4.1-Fr.4.6.

[0017] (6) Obtaining japonidiene D: The subfraction Fr.4.5 obtained by elution with 70% methanol-water was separated and purified by gel chromatography to prepare the polyketide compound japonidiene D.

[0018] As a preferred technical solution, the composition and formulation of the PDA plate culture medium in step (1) are as follows: 6g potato starch, 20g agar, 20g glucose and 1L water.

[0019] As a preferred technical solution, the volume ratio of ethyl acetate added in step (3) to the volume of potato glucose aqueous culture medium is 1:1; the power of ultrasonic extraction is 800W and the time is 30min; the vacuum degree of vacuum concentration is 0.08-0.10Mpa.

[0020] As a preferred technical solution, in step (4), the volume ratio of methanol to crude extract is 1:1, the particle size of silica gel is 100-200 mesh, and the mass ratio of crude extract to silica gel is 1:1; the vacuum degree of the reduced pressure vacuum is 0.05-0.08 MPa.

[0021] As a preferred technical solution, the application of the polyketide compound japonicine D in inhibiting the germination of seeds of the weed Amaranthus retroflexus is discussed.

[0022] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The polyketide compound japonidiene D, derived from the tobacco endophytic fungus Aspergillus japonicus TE-739D, was prepared by SciFinder. n According to database searches and comparisons with currently reported compounds, the compound japonidiene D is a novel compound with a newly reported structure.

[0024] (2) The preparation method provided by the present invention can efficiently separate and prepare the polyketide compound japonicine D;

[0025] (3) The polyketide compound japonicine D has a strong inhibitory effect on the seed germination of Amaranthus retroflexus, which is superior to the positive control drug glyphosate. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 Here is the molecular structure diagram of the polyketide compound japonicine D.

[0028] Figure 2 Here is the high-resolution mass spectrum of the polyketide compound japonicine D.

[0029] Figure 3 Here is the 1H NMR spectrum (600MHz, DMSO-d6) of the polyketide compound japoniciene D.

[0030] Figure 4 Here is the carbon spectrum (150 MHz, DMSO-d6) of the polyketide compound japoniciene D. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The Aspergillus japonicus TE-739D used in this embodiment of the invention was deposited on October 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.40901 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Potato Dextrose Water was purchased from Haibo Biotechnology, product number HB0233-4.

[0034] Example 1

[0035] The preparation method of the polyketide compound japoniciene D includes the following steps:

[0036] (1) Activation and expansion: In a sterile operating table, the mycelia of activated Aspergillus japonicus TE-739D were picked and inoculated onto PDA plate medium (6g potato extract powder, 20g agar, 20g glucose and 1L water) and cultured in a constant temperature incubator at 28℃ for 5 days.

[0037] (2) Fermentation culture: Cut off the bacterial block (1cm×1cm) from the above PDA plate and transfer it to a 1L Erlenmeyer flask containing 300mL potato glucose water culture medium. The culture medium has been sterilized at high temperature (121℃, 20min). Place it under normal temperature and light / dark conditions of 12h / 12h and let it ferment for 30 days.

[0038] (3) Preparation of crude extract: The culture medium after fermentation in step (2) was extracted with ethyl acetate (300 mL / bottle) by ultrasonic extraction (ultrasonic power 800 W, ultrasonic working time 30 min), and concentrated under vacuum (vacuum degree 0.09 Mpa) to prepare crude extract.

[0039] (4) Initial separation of components: 20 mL of the above crude extract was dissolved in 20 mL of methanol, and then 100-200 mesh silica gel was added and mixed (the mass ratio of crude extract to silica gel was 1:1). Then, the components were separated by silica gel column chromatography under reduced pressure (vacuum degree 0.05 MPa). 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, and methanol-dichloromethane mixtures with volume ratios of 10:90, 20:80 and 30:70 were used for elution. Similar components were combined by thin-layer chromatography to obtain 7 components Fr.1-Fr.7 with increasing polarity.

[0040] (5) Component reseparation: Component Fr.4, which was eluted by a mixture of petroleum ether and ethyl acetate at a volume ratio of 50:50, was further eluted by gradient chromatography using a LobarLiChroprep RP-18 reversed silica gel column with methanol-water at volume ratios of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. After analysis by thin-layer chromatography (TLC), similar components were combined to obtain six sub-components Fr.4.1-4.6.

[0041] (6) Obtaining japonidiene D: The subfraction Fr.4.5 obtained by elution with 70% methanol-water was separated by Sephadex LH-20 gel chromatography column and purified by semi-preparative high performance liquid chromatography (55% acetonitrile-water) to finally obtain the polyketide compound japonidiene D.

[0042] The molecular structure of the polyketide compound japonidiene D was determined by high-resolution mass spectrometry and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy. The experimental results are as follows: Figure 1 As shown, the high-resolution mass spectrum of the polyketide compound japoniciene D is as follows: Figure 2 As shown; the 1H NMR spectrum (600MHz, DMSO-d6) of the polyketide compound japoniciene D is shown below. Figure 3 As shown; the carbon spectrum (150MHz, DMSO-d6) of the polyketide compound japoniciene D is shown below. Figure 4 As shown.

[0043] The physicochemical properties of the polyketide compound japoniciene D are as follows:

[0044] Properties: Pale yellow amorphous solid; Solubility: Easily soluble in DMSO and methanol; Molecular formula: C 22 H 28 O6; Specific rotation: [α] 25 D+12.5 (c=0.04, MeOH); UV absorption spectrum λ max 238, 275 nm; High-resolution mass spectrometry (HRESI-MS): m / z 389.1965 [M+H] + (Theoretical value C) 22 H 29 O6 + (389.1959); the proton and carbon NMR data are shown in Table 1.

[0045] Table 1. NMR data (DMSO-d6) of the polyketide compound japonicine D.

[0046]

[0047] Example 2

[0048] This embodiment uses a seed germination inhibition method to determine the inhibitory activity of the polyketide compound japonidiene D on the seed germination of Amaranthus retroflexus. The specific process is as follows:

[0049] (1) Seed germination: Select uniform-sized and plump Amaranthus retroflexus seeds (provided by the Marine Agriculture Research Center of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences) and soak them in sterile water for 8 hours.

[0050] (2) Seed disinfection: Soak the above-soaked Amaranthus retroflexus seeds in a 3% sodium hypochlorite solution for 15 minutes, and rinse them repeatedly with sterile water three times.

[0051] (3) Preparation of test reagents:

[0052] Preparation of stock solution containing the polyketide compound japonidiene D: Weigh 0.3 mg of the polyketide compound japonidiene D and add it to 1.5 mL of methanol solution to prepare a stock solution with a concentration of 0.2 mg / mL;

[0053] Preparation of stock solution containing glyphosate (positive control drug): Weigh 0.3 mg of glyphosate and add it to 1.5 mL of sterile water to prepare a stock solution with a concentration of 0.2 mg / mL;

[0054] (4) Activity test: In a clean bench, sterilized filter paper was placed into a 12-well plate and divided into the following groups for the experiment:

[0055] Treatment group 1: 300 μL of mother liquor of polyketide compound japonicine D (dissolved in methanol and dried) + 300 μL of sterile water (the methanol has been dried, so the total solvent is actually 300 μL);

[0056] Control group: 300 μL sterile water;

[0057] Treatment group 2: 300 μL glyphosate stock solution (positive control);

[0058] Each group was set up with 3 replicates, with 10 seeds in each replicate. After sealing, the seeds were placed in an artificial incubator and cultured for 96 hours. The root shoot length of the seeds was measured, and the inhibition rates of the polyketide compound japonidiene D and glyphosate on the germination of Amaranthus retroflexus seeds were calculated. The experimental results are shown in Table 2.

[0059] The calculation formula is:

[0060] Bud (root) inhibition rate (%) = [Bud ​​(root) length of blank control group - Bud (root) length of treatment group] / Bud (root) length of blank control group × 100%.

[0061] Table 2. Inhibitory activity of the polyketide compound japonicidiene D on seed germination of Amaranthus retroflexus (0.2 mg / mL)

[0062]

[0063] Note: Data in the table are mean ± standard error.

[0064] Results analysis: As shown in Table 2, the polyketide compound japoniciene D has a strong inhibitory activity on the germination of Amaranthus retroflexus seeds. At a concentration of 0.2 mg / mL, the inhibition rate of shoot growth of Amaranthus retroflexus seeds was 64.15%, which was better than the positive control herbicide glyphosate at 45.08%.

[0065] Example 3

[0066] A strain of Aspergillus japonicus TE-739D is characterized by being deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.40901.

[0067] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polyketide compound, japonidiene D, characterized by, The molecular formula of the polyketide japonidiene D is C 22 H 28 O6, as shown in the following structural formula:

2. Use of Aspergillus japonicus TE-739D in the preparation of a polyketide compound, japonidiene D, characterized in that, The Aspergillus japonicus TE-739D is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.40901.

3. The method of producing the polyketide japonidiene D according to claim 1, characterized by, The method comprises the following steps: (1) Subculture: inoculate the mycelium of Aspergillus japonicus TE-739D into PDA plate culture medium, and subculture at 28°C for 3-5 days; (2) Fermentation culture: cut the mycelium on the PDA plate culture medium in step (1), and transfer to potato glucose water culture medium, and place under the condition of normal temperature and light / darkness of 12h / 12h, and stand for fermentation for 30-35 days; (3) Preparation of crude extract: after fermentation in step (2), the culture medium is extracted by ultrasonic extraction with ethyl acetate, and concentrated under vacuum and reduced pressure to prepare a crude extract; (4) Initial separation of components: after the crude extract in step (3) is dissolved with methanol, silica gel is added, and after mixing, the components are separated by vacuum silica gel column chromatography, and eluted with petroleum ether-ethyl acetate mixed solution with a volume ratio of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70 and 0:100, and methanol-dichloromethane mixed solution with a volume ratio of 10:90, 20:80 and 30:70, and similar components are combined by thin layer chromatography to obtain seven components Fr.1-Fr.7 with different polarities from small to large; (5) Re-separation of components: the component Fr.4 eluted with the petroleum ether-ethyl acetate mixed solution with a volume ratio of 50:50 in step (4) is gradient eluted by reverse silica gel column chromatography, and the elution system is methanol-water with a volume ratio of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, and the eluted components are detected by thin layer chromatography, and similar components are combined to obtain six sub-components Fr.4.1-Fr.4.6; (6) Obtaining of japonidiene D: the sub-component Fr.4.5 eluted with the elution system of 70% methanol-water is separated and purified by gel chromatography column to prepare the polyketide compound japonidiene D.

4. The method of producing polyketide japonidiene D according to claim 3, characterized by, The PDA plate culture medium in step (1) comprises the following components and is prepared in the following ratio: potato powder 6g, agar 20g, glucose 20g and water 1L.

5. The method of producing polyketide japonidiene D according to claim 3, characterized by, The volume ratio of the added ethyl acetate to the potato glucose water culture medium in step (3) is 1:1, the ultrasonic extraction power is 800W, and the ultrasonic extraction time is 30min, and the vacuum degree of the vacuum and reduced pressure concentration is 0.08-0.10Mpa.

6. The method of producing polyketide japonidiene D according to claim 3, characterized by, The volume ratio of the methanol to the crude extract in step (4) is 1:1, the particle size of the silica gel is 100-200 meshes, the mass ratio of the crude extract to the silica gel is 1:1, and the vacuum degree of the vacuum and reduced pressure is 0.05-0.08Mpa.

7. Use of the polyketide compound japonidiene D according to claim 1 or the polyketide compound japonidiene D prepared according to any one of claims 3 to 6 for inhibiting the seed germination of the weed Amaranthus retroflexus.

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