A fungal-derived Japoninolide E compound, its preparation method and application

Abstract

The application discloses a fungus-derived Japoninolide E compound as well as a preparation method and application thereof, and belongs to the technical field of natural product chemistry and agricultural bioactivity evaluation. Aspergillus japonicus 334is separated from a fermentation product, and the structure identification is based on high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Under a test concentration, the Japoninolide E has certain inhibitory effects on the root and bud growth of amaranthus retroflexus and axonopus compressus seeds, and therefore, the Japoninolide E belongs to a fungus metabolic product with a potential herbicidal activity.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of natural product chemistry and agricultural bioactivity evaluation, and more specifically to the method for obtaining Japoninolide E compound, its structural identification data, and its use as an active ingredient for inhibiting weed seed germination. Background Technology

[0002] Green agricultural production requires herbicidal active substances to possess both effectiveness and ecological compatibility. Microbial fermentation products can be obtained through controlled culture and often contain natural products with unique skeletons, making them suitable as an important resource for discovering novel herbicidal active structures. Chinese invention patent CN119791126B discloses a herbicide derived from *Gastrodia elata* (…). Dactylobotrys graminicola 4,15-Diacetylverrucarol, a trichothecene compound, exhibits significant growth inhibition against barnyardgrass and crabgrass. At a concentration of 0.1 g / L, the fresh weight inhibition rates are 92.87% and 91.41%, respectively. However, its fresh weight inhibition rate against soybean is only 3.24%, with a selectivity index of 2.67, indicating relative safety for soybeans. This compound shows excellent activity against gramineous weeds but is largely ineffective against broadleaf weeds, suggesting its potential as a candidate target for selective control of gramineous weeds. Another Chinese invention patent, CN120167443A, discloses Phyllanthusmin C, a compound derived from *Taraxacum officinale*. This patent demonstrates that Phyllanthusmin C exhibits good root length and fresh weight inhibition against common farmland gramineous weeds such as barnyardgrass, goosegrass, crabgrass, foxtail, and *Symplocos edulis*, as well as sedges. At the same concentration, its inhibition rate is comparable to or even better than that of the control herbicide glyphosate, demonstrating excellent inhibitory activity against grass and sedge weeds.

[0003] In the current research field of herbicidal active natural products, although a large number of secondary metabolites with herbicidal activity have been reported, compounds that simultaneously meet the following three conditions are still relatively scarce: First, they have clear information on the strain origin, including traceable systematic taxonomic data such as strain identification, preservation number, and fermentation conditions; second, they have a complete isolation and purification process, covering extraction, chromatographic separation, and structural identification, to ensure clear origin and confirmed structure of the compound; and third, they provide comparable quantitative activity data (such as inhibition rate, IC50, etc.). 50 Value, EC 50 The complete presentation of these three types of information (such as herbicidal activity value) is crucial for supporting an objective evaluation of their herbicidal activity and practical application potential. Therefore, the systematic accumulation of such high-quality compounds remains of significant research value and practical importance.

[0004] Therefore, providing a fungal-derived Japoninolide E compound, its preparation method, and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a fungal-derived Japoninolide E compound, its preparation method, and its application.

[0006] The compound Japoninolide E is derived from Aspergillus japonicus 334, and its biological activity has been verified through a seed germination model.

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

[0008] A fungal compound, Japoninolide E, has the following molecular structure: .

[0009] Furthermore, the aforementioned novel compound Japoninolide E was synthesized through... Aspergillus japonicus The compound was isolated from the fermentation product of 334, and its structure was identified using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Table 1 lists the compounds in DMSO- d NMR data in 6.

[0010] Furthermore, the above-mentioned compound Japoninolide E was used to inhibit the germination of seeds of Amaranthus retroflexus and Eleusine indica.

[0011] Furthermore, the activity assay of Japoninolide E was performed using the following seed germination system: (1) After screening, the test seeds were soaked in sterile water for 8 hours to ensure uniform water absorption; then they were soaked in 3% sodium hypochlorite for 15 minutes and rinsed three times with sterile water before being used for germination experiments.

[0012] (2) Japoninolide E was prepared into a sample solution of 0.2 mg / mL by adding 0.3 mg of Japoninolide E to 1.5 mL of methanol, and the Japoninolide E stock solution was obtained; glyphosate was prepared into a positive control at the same concentration, and the glyphosate stock solution was obtained.

[0013] (3) In a clean bench, sterilized filter paper was placed into 12-well plates. For the Japoninolide E group, 300 μL of Japoninolide E stock solution was added to each well, and the methanol solvent was allowed to dry (3 h). In the glyphosate positive control group, 300 μL of glyphosate stock solution was added to each well. The Japoninolide C group and the glyphosate positive control group were the treatment groups. In the blank group, 300 μL of sterile water was added to each well. Each treatment was repeated 3 times, with 10 seeds placed in each well. After sealing, the plates were placed in a 28℃ incubator for 96 h. The root and shoot lengths of the seeds were measured, and the inhibition rates of Japoninolide E and glyphosate on the germination of the two weed seeds were calculated. The calculation formula is as follows: Bud (root) inhibition rate (%) = [Bud ​​(root) length of blank group] Treatment bud (root) length / control group bud (root) length × 100%.

[0014] Aspergillus japonicum Aspergillus japonicus 334 See literature: Dong, C.; Wang, H.; Xu, K.;Gu, G.; Zhang, P. LC-MS / MS-Guided Discovery of Herbicidal Himeic AcidDerivatives from the Nicotiana tabacum-Derived Fungus Aspergillus japonicus 334. J. Agric. Food Chem. 2025, 73, 21463 21472. Aspergillus japonicum 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.

[0015] The compound Japoninolide E has a clear fungal fermentation origin and a workable isolation method, and its inhibitory results on weed seed growth indicators provide a supplement to the screening of natural herbicidal active substances.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a fungal-derived Japoninolide E compound, its preparation method and application. At the tested concentration, Japoninolide E has a certain inhibitory effect on the root and shoot growth of Amaranthus retroflexus and Eleusine indica seeds, indicating that it belongs to a fungal metabolite with potential herbicidal activity. Attached Figure Description

[0017] 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.

[0018] Figure 1 Here is the molecular structural formula of the Japoninolide E compound.

[0019] Figure 2 This is a high-resolution mass spectrum of the Japoninolide E compound.

[0020] Figure 3 The 1H NMR spectrum of the Japoninolide E compound (600 MHz, DMSO- d 6) Figure.

[0021] Figure 4 Carbon spectroscopy (150 MHz, DMSO-) of Japoninolide E compound d 6) Figure. Detailed Implementation

[0022] 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.

[0023] To more clearly illustrate the technical effects of Japoninolide E, the following examples of structural characterization and activity determination are provided.

[0024] Example 1 1) The following steps are used to prepare compound Japoninolide E: (1) Aspergillus japonicus After activating and culturing 334 on PDA plates to obtain mycelia in a uniform state, approximately 1 cm × 1 cm mycelial blocks were used as inoculum for liquid fermentation.

[0025] (2) The inoculum was transferred into sterilized PDB medium and fermented for 30 days under static conditions at room temperature. The fermented product was extracted by ultrasonic extraction with ethyl acetate and concentrated under reduced pressure to obtain crude extract.

[0026] (3) After dissolving the crude extract in methanol and mixing it with silica gel, silica gel column chromatography was performed. First, a gradient elution of petroleum ether-ethyl acetate was performed at 100:1, 50:1, 25:1, 10:1, 5:1, and 2:1. Then, a gradient elution of methanol-dichloromethane was performed at 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1. Based on the TLC results, similar eluted fractions were combined to obtain Fr.1-Fr.7. (4) The Fr.4 eluent obtained in step (3) by petroleum ether-ethyl acetate at volume ratios of 10:1 and 5:1 was eluted with a methanol-water gradient on a Lobar LiChroprep RP-18 column: from 30% to 100% methanol. Based on the TLC and HPLC results, similar eluents were combined and separated to obtain 5 subfractions Fr.4.1-4.5; the subfraction containing Japoninolide E was Fr.4.1, which corresponds to an elution ratio of 55% methanol-water system; (5) Subfraction Fr.4.1 (elution system: 55% methanol-water) was further purified by Sephadex LH-20 gel column chromatography with a mobile phase of dichloromethane-methanol (v / v) of 1:1 and semi-preparative HPLC with a v / v ratio of 55:45. The target peak was collected and the solvent was removed to obtain Japoninolide E, whose molecular structure is shown in the figure. Figure 1 .

[0027] 2) Structural confirmation of compound Japoninolide E. The separated compound was analyzed by high-resolution mass spectrometry and one-dimensional nuclear magnetic resonance spectroscopy. The physicochemical properties of compound Japoninolide E are as follows: Appearance: Pale yellow amorphous solid; α +74 ( c 0.25, MeOH); UV absorption spectrum λ_max (log ε 287 (2.67) nm; Electronic Circular Dichroism (EMD) λ (Δ ε ) 216 ( 1.06), 250 (+3.00), 289 (+1.78) nm; High-resolution mass spectrometry (HRESIMS) m / z 473.1941 [M + Na] + (The calculated value is C) 27 H 30 O6Na + ,473.1935). The 1H NMR spectrum of compound Japoninolide E ( 1H NMR data and carbon spectra ( 13 The C NMR data are shown in Table 1.

[0028] Table 1: NMR data of compound Japoninolide E ( 1 H: 600 MHz; 13 C: 150 MHz; Test solvent: DMSO- d 6)

[0029] The high-resolution mass spectrum of the Japoninolide E compound is shown below. Figure 2 The proton NMR spectrum of the Japoninolide E compound (600 MHz, DMSO- d 6) See diagram Figure 3 Carbon-150 MHz, DMSO- (C-NMR spectrum of Japoninolide E compound) d 6) See diagram Figure 4 .

[0030] Example 2: Inhibitory effect of Japoninolide E compound on seed germination of Amaranthus retroflexus and Eleusine indica The method for determining the inhibition of seed germination by Japoninolide E 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.

[0031] Seed disinfection: Soak the soaked weed seeds in a 3% sodium hypochlorite solution for 15 minutes, and rinse them repeatedly with sterile water three times.

[0032] (2) Preparation of test reagents Preparation of stock solution containing compound Japoninolide E: Weigh 0.3 mg of compound Japoninolide E and add it to 1.5 mL of methanol to prepare a stock solution with a concentration of 0.2 mg / mL.

[0033] 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.

[0034] (3) Activity test In a clean bench, sterilized filter paper was placed into 12-well plates. For the Japoninolide E group, 300 μL of stock solution containing Japoninolide E was added to each well, and the plates were allowed to stand until the methanol solvent was evaporated (3 h). In the glyphosate positive control group, 300 μL of stock solution containing glyphosate was added to each well. The Japoninolide C group and the glyphosate positive control group were the treatment groups. In the blank group, 300 μL of sterile water was added to each well. Each treatment was replicated three times, with 10 seeds placed in each well. After sealing, the plates were incubated at 28°C for 96 h. The root and shoot lengths of the seeds were measured, and the inhibition rates of Japoninolide E and glyphosate on the germination of the two weed seeds were calculated. The calculation formula is as follows: Bud (root) inhibition rate (%) = [Bud ​​(root) length of blank group] Treatment bud (root) length / control group bud (root) length × 100%.

[0035] The inhibitory activity of compound Japoninolide E on the seed germination of Amaranthus retroflexus and Eleusine indica is shown in Table 2.

[0036] Table 2: Inhibitory activity of compound Japoninolide E on seed germination of Amaranthus retroflexus and Eleusine indica (0.2 mg / mL)

[0037] Note: The data in the table are mean ± standard error.

[0038] Table 2 shows that Japoninolide E exhibits measurable inhibitory effects on the root and shoot growth of both Amaranthus retroflexus and Eleusine indica. Although there are differences in activity intensity among different indicators, it still demonstrates the screening value of natural product herbicidal activity.

[0039] 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 fungal-derived Japoninolide E compound, characterized in that, Its molecular structure is as follows: 。 2. The method for preparing the Japoninolide E compound according to claim 1, characterized in that, Includes the following steps: (1) Aspergillus japonicus After activating and culturing 334 on a PDA plate, the fermentation inoculum was collected; (2) The inoculum was transferred into sterilized PDB medium and fermented at room temperature for 30 days. The fermentation product was extracted with ethyl acetate by ultrasonic extraction and concentrated under reduced pressure to obtain crude extract. (3) After dissolving the crude extract in methanol and mixing it with silica gel, silica gel column chromatography was performed. First, a gradient elution of petroleum ether-ethyl acetate was performed at 100:1, 50:1, 25:1, 10:1, 5:1, and 2:

1. Then, a gradient elution of methanol-dichloromethane was performed at 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, and 0:

1. Based on the TLC results, similar eluted fractions were combined to obtain Fr.1-Fr.

7. (4) The Fr.4 eluent obtained in step (3) by petroleum ether-ethyl acetate at volume ratios of 10:1 and 5:1 was eluted with a methanol-water gradient on a Lobar LiChroprep RP-18 column: from 30% to 100% methanol. Based on the TLC and HPLC results, similar eluents were combined and separated to obtain 5 subfractions Fr.4.1-4.5; the subfraction containing Japoninolide E was Fr.4.1, which corresponds to an elution ratio of 55% methanol-water system; (5) Subfraction Fr.4.1 was further purified by Sephadex LH-20 gel column with dichloromethane-methanol as mobile phase (1:1, volume ratio) and semi-preparative HPLC (55:45, volume ratio). The target peak was collected and the solvent was removed to obtain Japoninolide E.

3. The application of the Japoninolide E compound according to claim 1 in inhibiting the germination of seeds of Amaranthus retroflexus and Eleusine indica.

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