A compound Japoninolide D, its preparation method and applications

Abstract

This invention discloses a compound Japoninolide D, its preparation method, and its applications, belonging to the field of microbial metabolite technology. The Japoninolide D compound is derived from... Aspergillus japonicus Japoninolide D was isolated from the fermentation product of 334. Bioactivity assays showed that it significantly inhibited the seed germination of *Amaranthus retroflexus* and *Eleusine indica*, particularly exhibiting a high inhibition rate on the root and shoot growth of *Amaranthus retroflexus*. It can be used to prepare herbicides that inhibit weed seed germination. This invention provides a novel lead compound for the development of new herbicides and has promising application prospects.

Description

Technical Field

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

[0002] Agricultural weed control has long faced the dual challenges of homogeneous herbicides and the overlapping risks of resistance. Existing herbicides have simple structures and limited targets; coupled with long-term, extensive use, many weeds have developed resistance to mainstream herbicides, making control increasingly difficult. Against this backdrop, discovering herbicidal active molecules with novel structures based on natural products can help expand the herbicide lead compound library, enrich the structural types of compounds, and provide a high-quality chemical basis for subsequent research on mechanisms of action.

[0003] Chinese invention patent CN122036657A discloses a furanone compound derived from the fungus *Aspergillus* sp. This compound exhibits significant inhibitory effects on common weeds such as foxtail grass and amaranth, with the inhibitory effect increasing with increasing concentration. Activity assays showed that amaranth exhibited a concentration-dependent growth-inhibiting effect, mainly manifested as leaf shrinkage and plant dwarfing; while foxtail grass showed distinctly different response characteristics at different concentrations: it exhibited a growth-promoting effect at sublethal concentrations (≤100 μg / mL), while showing a significant growth-inhibiting effect at critical concentrations (200 μg / mL). This concentration-dependent response difference provides important clues for further understanding the characteristics of this compound's action.

[0004] Dimeric sesquiterpene lactones, due to their unique ring skeleton and physicochemical properties, can specifically bind to key enzymes or receptors in plants, thus exhibiting excellent herbicidal activity and other agricultural application potential. These compounds possess outstanding advantages such as high efficiency, low toxicity, crop safety, and low likelihood of inducing herbicide resistance in weeds. Therefore, in-depth research on the extraction and preparation methods of dimeric sesquiterpene lactones and systematic evaluation of their herbicidal bioactivity have significant scientific research value and application prospects.

[0005] Currently, there are relatively few types of naturally derived dimeric sesquiterpene lactones with herbicidal properties, resulting in limited selectivity.

[0006] Therefore, how to enrich the variety of 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

[0007] In view of this, the present invention has successfully extracted and prepared a type of Aspergillus japonicus. Aspergillus japonicusThe compound Japoninolide D, derived from 334, was found to have inhibitory activity against the germination of seeds of Amaranthus retroflexus and Eleusine indica, providing technical support for the further development of biopesticides.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A compound, Japoninolide D, has the following structural formula: .

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned compound Japoninolide D, comprising the following steps: (1) Aspergillus japonicus Aspergillus japonicus After activation with 334, the mycelium was inoculated onto PDA plates and cultured at 28°C until the mycelium had fully grown. (2) Cut off the bacterial blocks from the PDA plate culture medium in step (1) and transfer them to potato glucose water culture medium. Ferment for 30 days under the conditions of standing at room temperature and alternating light and dark for 12 hours each. (3) After fermentation, ethyl acetate was added for ultrasonic extraction. The extract was concentrated under reduced pressure to prepare a crude extract containing a variety of metabolites. (4) The crude extract was dissolved in an equal volume of methanol and then mixed with 100-200 mesh silica gel at a mass ratio of 1:1. The mixture was subjected to reduced pressure silica gel column chromatography under a vacuum of 0.05 MPa. The mixture was eluted sequentially with petroleum ether-ethyl acetate mixtures with volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, and 2:1 and methanol-dichloromethane mixtures with volume ratios of 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1. Similar eluted fractions were combined according to the TLC results to obtain Fr.1-Fr.7. (5) The fraction Fr.4 eluted from the petroleum ether-ethyl acetate 10:1 and 5:1 mixtures was subjected to Lobar LiChroprep RP-18 reversed-phase column chromatography with gradient elution of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% methanol-water systems. Based on the TLC and HPLC results, similar eluents were combined and separated to obtain 5 subfractions Fr.4.1-4.5. (6) The sub-component Fr.4.2 was first separated by Sephadex LH-20 gel chromatography with dichloromethane-methanol as the mobile phase at a volume ratio of 1:1, and then purified by semi-preparative high performance liquid chromatography with methanol-water as the mobile phase at a volume ratio of 40:60. The target peak was collected and the solvent was removed to obtain Japoninolide D.

[0011] As a preferred technical solution, the composition and formulation of the PDA plate culture medium in step (1) are as follows: 6 g potato starch, 20 g agar, 20 g glucose and 1 L water.

[0012] As a preferred technical solution, the volume ratio of ethyl acetate added in step (3) to potato glucose water culture medium is 1:1; the power of ultrasonic extraction is 800 W and the time is 30 min.

[0013] One object of this application is to provide the use of the compound Japoninolide D or the compound Japoninolide D prepared by the method in inhibiting the germination of seeds of the weeds Amaranthus retroflexus and / or Eleusine indica.

[0014] One object of this application is to provide the use of the compound Japoninolide D or the compound Japoninolide D prepared by the method in the preparation of a herbicide for inhibiting the germination of seeds of the weeds Amaranthus retroflexus and / or Eleusine indica.

[0015] One object of this application is to provide: a herbicide comprising the Japoninolide D compound or an agriculturally acceptable salt thereof, and an agriculturally acceptable carrier and / or adjuvant.

[0016] One object of this application is to provide: the aforementioned Aspergillus japonicus Aspergillus japonicus Application of 334 in the preparation of compound Japoninolide D.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first from Aspergillus japonicus Japoninolide D was isolated from the fermentation products of 334, enriching the compound library and providing a novel lead compound for the creation of new pesticides.

[0018] (2) The preparation method provided by the present invention is simple to operate, has good reproducibility, and can obtain Japoninolide D samples with high purity, which is suitable for large-scale preparation.

[0019] (3) This invention is the first to discover that Japoninolide D has an inhibitory effect on the germination of seeds of Amaranthus retroflexus and Eleusine indica, and can be further developed as a herbicidal active compound of microbial origin.

[0020] (4) Japoninolide D is derived from natural fungi, is environmentally friendly, and is in line with the development direction of green pesticides. Attached Figure Description

[0021] 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.

[0022] Figure 1 Here is the molecular structure diagram of compound Japoninolide D.

[0023] Figure 2 Here is the high-resolution mass spectrum of compound Japoninolide D.

[0024] Figure 3 For example: the 1H NMR spectrum of compound Japoninolide D (600 MHz, DMSO- d 6) Figure.

[0025] Figure 4 For example: Carbon spectrum of compound Japoninolide D (150 MHz, DMSO- d 6) Figure. Detailed Implementation

[0026] 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.

[0027] Aspergillus japonicus used in the embodiments of the present invention Aspergillus japonicus Strains 334 are previously published strains; see the following reference: Dong, C.; Wang, H.; Xu, K.; Gu, G.; Zhang, P. LC-MS / MS-Guided Discovery of Herbicidal Himeic Acid Derivatives from the Nicotiana tabacum-Derived Fungus Aspergillus japonicus 334. J. Agric. Food Chem. 2025, 73, 21463 21472. Aspergillus japonicus 334 is currently held by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences and can be obtained from the applicant or the inventor team.

[0028] Potato Dextrose Water, used for fungal fermentation culture, was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., with catalog number HB0233-4.

[0029] The composition and ratio of PDA plate medium are as follows: 6 g potato extract powder, 20 g glucose, 20 g agar, and water to a final volume of 1 L. Weigh each component according to the ratio, dissolve by heating, dispense into containers, autoclave at 121℃ for 15 min, cool to approximately 50℃, and then pour into plates for later use.

[0030] Example 1 The preparation method of compound Japoninolide D includes the following steps: (1) Aspergillus japonicus Aspergillus japonicus After activation with 334, the mycelium was inoculated onto PDA plates and cultured at 28°C until the mycelium had fully grown. (2) Cut off the bacterial blocks from the PDA plate culture medium in step (1) and transfer them to potato glucose water culture medium. Ferment for 30 days under the conditions of standing at room temperature and alternating light and dark for 12 hours each. (3) After fermentation, ethyl acetate was added for ultrasonic extraction. The extract was concentrated under reduced pressure to prepare a crude extract containing a variety of metabolites. (4) Dissolve the crude extract in an equal volume of methanol, then mix it with 100-200 mesh silica gel at a mass ratio of 1:1. Perform vacuum silica gel column chromatography under a vacuum of 0.05 MPa. Elute sequentially with petroleum ether-ethyl acetate mixtures with volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, 2:1 and methanol-dichloromethane mixtures with volume ratios of 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1. Combine similar eluted fractions according to the TLC results to obtain Fr.1-Fr.7. (5) Fraction Fr.4, eluted from a 10:1 and a 5:1 mixture of petroleum ether and ethyl acetate, was subjected to reversed-phase column chromatography on a Lobar LiChroprep RP-18 with gradient elutions of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol-water systems. Based on TLC and HPLC results, similar eluents were combined to obtain five subfractions Fr.4.1-4.5. (The subfraction containing Japoninolide D was Fr.4.2, corresponding to an elution ratio of 40% methanol-water system). (6) The sub-fraction Fr.4.2 (elution system of 40% methanol-water) was first separated by Sephadex LH-20 gel chromatography with dichloromethane-methanol as the mobile phase at a volume ratio of 1:1, and then purified by semi-preparative high performance liquid chromatography with methanol-water as the mobile phase at a volume ratio of 40:60. The target peak was collected and the solvent was removed to obtain Japoninolide D.

[0031] The molecular structure of the isolated compound Japoninolide D was determined by high-resolution mass spectrometry and one-dimensional nuclear magnetic resonance spectroscopy. The experimental results are as follows: Figure 1 As shown; the high-resolution mass spectrum of compound Japoninolide D is as follows. Figure 2 As shown; the 1H NMR spectrum of compound Japoninolide D (600 MHz, DMSO- d 6) As shown in the figure Figure 3 As shown; the carbon spectrum of compound Japoninolide D (150 MHz, DMSO- d 6) As shown in the figure Figure 4 As shown.

[0032] The physicochemical properties of compound Japoninolide D are as follows: Properties: Pale yellow amorphous solid; Specific rotation: [α]+65 ( c 0.12, MeOH); UV absorption spectrum λ_max (log ε 285 (2.74) nm; Electronic Circular Dichroism (EMD) λ (Δ ε ) 213 ( 2.18), 250 (+3.17), 286 (+1.88) nm; High-resolution mass spectrometry (HRESIMS) m / z 473.1941 [M + Na] + (Calculated value is C) 27 H 30 O6Na + ,473.1935). Its nuclear magnetic resonance hydrogen spectrum ( 1 H NMR data and carbon spectra ( 13 The C NMR data are shown in Table 1.

[0033] Table 1: NMR data of the compound Japoninolide D

[0034] Example 2 This embodiment uses a seed germination inhibition method to determine the inhibitory activity of compound Japoninolide D on the seed germination of Amaranthus retroflexus and Eleusine indica. The specific process is as follows: (1) Seed preparation process Seed germination: Select two types of weed seeds (Amaranthus retroflexus and Eleusine indica, provided by the Marine Agriculture Research Center of the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences) that are uniform in size and plump, and soak them in sterile water for 8 hours.

[0035] (2) Seed disinfection: Soak the above-mentioned soaked weed seeds in a 3% sodium hypochlorite solution for 15 minutes, and rinse them repeatedly with sterile water three times.

[0036] (3) Preparation of test reagents Preparation of stock solution: Weigh 0.3 mg of compound Japoninolide D and add it to 1.5 mL of methanol solution to prepare a stock solution with a concentration of 0.2 mg / mL.

[0037] Preparation of stock solution for 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 of 0.2 mg / mL.

[0038] (4) Activity test In a clean bench, sterilized filter paper was placed into 12-well plates. For the treatment group, 300 μL of stock solution containing Japoninolide D was added to each well, and the plates were allowed to stand until the methanol solvent was evaporated (3 h). In the treatment and control groups, 300 μL of sterile water was added to each well; in the control group, 300 μL of glyphosate stock solution was added to each well. All groups were replicated in triplicate, with 10 seeds in each replicate. After sealing, the plates were incubated in an artificial incubator for 96 h. The root and shoot lengths of the seeds were measured, and the inhibition rates of Japoninolide D and glyphosate on the germination of the two weed seeds were calculated. The experimental results are shown in Table 2.

[0039] The calculation formula is: Bud (root) inhibition rate (%) = [Bud ​​(root) length of blank control group] [Treatment bud (root) length] / [Blank control bud (root) length] × 100% Table 2: Inhibitory activity of compound Japoninolide D on seed germination of Amaranthus retroflexus and Eleusine indica (0.2 mg / mL)

[0040] Note: Data in the table are mean ± standard error. Results analysis: The results showed that Japoninolide D had a significant effect on the seed germination model of Amaranthus retroflexus, with root and shoot inhibition rates of 63.12% and 76.80%, respectively, indicating that it has a good inhibitory effect on the early growth of Amaranthus retroflexus.

[0041] 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.

[0042] 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 compound Japoninolide D, characterized in that, The structural formula of the compound Japoninolide D is as follows: 。 2. The method for preparing the compound Japoninolide D according to claim 1, characterized in that, Includes the following steps: (1) Aspergillus japonicus Aspergillus japonicus After activation with 334, the mycelium was inoculated onto PDA plates and cultured at 28°C until the mycelium had fully grown. (2) Cut off the bacterial blocks from the PDA plate culture medium in step (1) and transfer them to potato glucose water culture medium. Ferment for 30 days under the conditions of standing at room temperature and alternating light and dark for 12 hours each. (3) After fermentation, ethyl acetate was added for ultrasonic extraction. The extract was concentrated under reduced pressure to prepare a crude extract containing a variety of metabolites. (4) Dissolve the crude extract in an equal volume of methanol, mix it with 100-200 mesh silica gel at a mass ratio of 1:1, and perform vacuum silica gel column chromatography under a vacuum of 0.05 MPa. Elute with petroleum ether-ethyl acetate mixtures with volume ratios of 100:1, 50:1, 25:1, 10:1, 5:1, 2:1 and methanol-dichloromethane mixtures with volume ratios of 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1, and combine similar eluted fractions according to the TLC results to obtain Fr.1-Fr.

7. (5) The fraction Fr.4 eluted from the petroleum ether-ethyl acetate 10:1 and 5:1 mixtures was subjected to Lobar LiChroprep RP-18 reversed-phase column chromatography with gradient elution of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% methanol-water systems. Based on the TLC and HPLC results, similar eluents were combined and separated to obtain 5 subfractions Fr.4.1-4.

5. (6) The sub-component Fr.4.2 was first separated by Sephadex LH-20 gel chromatography with dichloromethane-methanol as the mobile phase at a volume ratio of 1:1, and then purified by semi-preparative high performance liquid chromatography with methanol-water as the mobile phase at a volume ratio of 40:

60. The target peak was collected and the solvent was removed to obtain Japoninolide D.

3. The method for preparing compound Japoninolide D according to claim 2, characterized in that, The composition and formulation of the PDA plate culture medium in step (1) are as follows: 6 g potato starch, 20 g agar, 20 g glucose and 1 L water.

4. The method for preparing compound Japoninolide D according to claim 2, characterized in that, In step (3), the volume ratio of ethyl acetate to potato glucose aqueous culture medium is 1:1; the ultrasonic extraction power is 800 W and the time is 30 min.

5. The use of the compound Japoninolide D of claim 1 or the compound Japoninolide D prepared by the method of any one of claims 2-4 in inhibiting the germination of seeds of the weeds Amaranthus retroflexus and / or Eleusine indica.

6. The use of the compound Japoninolide D of claim 1 or the compound Japoninolide D prepared by the method of any one of claims 2-4 in the preparation of a herbicide for inhibiting the germination of seeds of the weeds Amaranthus retroflexus and / or Eleusine indica.

7. A herbicide, characterized in that, It comprises the Japoninolide D compound as described in claim 1 or an agriculturally acceptable salt thereof, as well as an agriculturally acceptable carrier and / or adjuvant.

8. Aspergillus japonicus in claim 3 Aspergillus japonicus Application of 334 in the preparation of compound Japoninolide D.

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