Compound containing polyhydronaphthalene skeleton and preparation method and application thereof

Abstract

The invention belongs to the technical field of phytochemistry, and particularly discloses a compound containing a polyhydronaphthalene skeleton and a preparation method and application thereof. The compound disclosed by the invention is prepared from a fermentation crude extract of a strain of tobacco endophytic fungus Aspergillus japonicus TE-739D, and the compound disclosed by the invention is prepared from the fermentation crude extract of the tobacco endophytic fungus Aspergillus japonicus TE-739D. Detailed structural identification is carried out on the compound through a modern spectroscopy technology, such as high-resolution mass spectrum HRESIMS and superconductive nuclear magnetic resonance spectrum NMR, and it is found that the compound contains a polyhydronaphthalene skeleton. Through SciFinderu database retrieval, it is found that the compound is a compound of a new structure, and the compound is named as japonidiene B. The compound japonidiene B has obvious herbicidal activity on amaranthus retroflexus and eleusine indica.

Description

Technical Field

[0001] This invention belongs to the field of phytochemistry technology, specifically relating to a compound containing a polyhydronaphthalene skeleton, its preparation method, and its application. Background Technology

[0002] Natural products have played a significant role in the development of green pesticides. Microbial-derived natural products, in particular, have been developed into products such as avermectin, jinggangmycin, and kasugamycin due to their novel structures and significant biological activities. These products are widely used in agricultural production and have yielded substantial economic and ecological benefits. Isolating and preparing highly active and pharmaceutically viable metabolites from microorganisms has become an important approach to green pesticide development. Chinese invention patent CN119490474A discloses a polyketide compound derived from *Trichoderma* sp. QTYC44, which can inhibit the growth of *Streptomyces streak*, *Staphylococcus aureus*, *Tetracoccus*, and *Actinidia kiwifruit* causal agent. Chinese invention patent CN117844671A discloses a soil actinomycete *Streptomyces violaceoruber*, whose fermented alkaloid compounds exhibit insecticidal activity against root-knot nematodes, achieving a 100% mortality rate at a concentration of 0.8 mg / mL. Chinese invention patent CN116903463A discloses a method for preparing and applying a β-bergamotane-type sesquiterpene. This compound is prepared by fermentation culture of *Pseudallescheria boydii*. Experiments have shown that this compound has inhibitory activity against agricultural pathogenic fungi and can be used as an inhibitor or fungicide for agricultural pathogens.

[0003] Natural products containing polyhydronaphthalene structures possess specific spatial configurations and chemical properties, enabling them to specifically interact with relevant enzymes and receptors in plants or animals, thereby exhibiting excellent agricultural and medicinal bioactivities. Due to the advantages of natural products such as high efficiency, safety, low toxicity, and crop-friendly properties, researching specific preparation methods and agricultural bioactivities of natural products containing polyhydronaphthalene skeletons is of great significance. Summary of the Invention

[0004] In view of this, the present invention has discovered a novel compound containing a polyhydronaphthalene skeleton from a strain of Aspergillus fungus, and for the first time reports a detailed preparation method and herbicidal activity of the compound. This compound has the potential to be developed into a green biological herbicide, providing a new compound template for the development of novel microbial natural product pesticides.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A compound containing a polyhydronaphthalene skeleton, the compound having the molecular formula C0 20 H 30 O6, the molecular structure is as follows:

[0007]

[0008] The present invention discloses a method for preparing the above-mentioned compound containing a polyhydronaphthalene skeleton, wherein Aspergillus japonicus TE-739D mycelium is selected for fermentation culture, and the fermentation broth is separated and purified.

[0009] Aspergillus japonicus TE-739D is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40901, classified as Aspergillus japonicus, on October 25, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Preferably, the specific steps include:

[0011] (1) Select the mycelium of Aspergillus japonicus TE-739D for fermentation culture to obtain fermentation broth;

[0012] (2) The fermentation broth was extracted with ethyl acetate and concentrated to obtain a crude extract.

[0013] (3) After mixing the crude extract with silica gel, gradient elution was performed by vacuum silica gel column chromatography.

[0014] (4) Collect the fraction Fr.5 eluted under the elution conditions of petroleum ether to ethyl acetate volume ratio of (0:10)-(3:7) and elute it through a reversed-phase silica gel column;

[0015] (5) The subfraction Fr.5.2 eluted under 40% methanol-water conditions was purified by gel column chromatography and semi-preparative high performance liquid chromatography to obtain the compound described above.

[0016] Preferably, the specific operation of step (1) is as follows: the mycelium of the activated strain Aspergillus japonicus TE-739D is picked and inoculated onto a PDA plate medium, cultured at 28°C for 5 days, the mycelium block on the PDA plate is cut and transferred to potato glucose water medium, cultured at room temperature for 30 days to obtain fermentation broth.

[0017] Preferably, the specific operation of step (2) is as follows: the fermentation broth in step (1) is extracted with ethyl acetate by ultrasonic extraction and concentrated under vacuum to obtain crude extract. The conditions for vacuum concentration are: vacuum degree 0.09 MPa, and concentration to a non-fluid state.

[0018] Preferably, the specific operation of step (3) is as follows: after dissolving the crude extract in methanol, 100-200 mesh silica gel is added and mixed, and then the components are separated by vacuum silica gel column chromatography under reduced pressure of 0.05 MPa. The components are eluted by a mixture of petroleum ether and ethyl acetate with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100, and a mixture of methanol and dichloromethane with volume ratios of 10:90, 20:80, and 30:70, respectively. Similar components are combined by thin-layer chromatography to obtain seven components Fr.1-Fr.7 with polarity from small to large.

[0019] Preferably, the specific operation of step (4) is as follows: the fraction Fr.5 eluted from the petroleum ether-ethyl acetate 30:70 mixed system and the 0:100 mixed system is subjected to gradient elution by LobarLiChroprep RP-18 reverse silica gel column chromatography, with the elution system being methanol-water volume ratios of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%, and the fractions are separated and combined to obtain 6 subfractions Fr.5.1-5.6.

[0020] Preferably, the subfraction Fr.5.2 obtained by elution system of 40% methanol-water is first separated by Sephadex LH-20 gel chromatography column and then purified by semi-preparative high performance liquid chromatography with mobile phase of 24% acetonitrile-water to obtain the compound.

[0021] The present invention also claims protection for the use of compounds containing a polyhydronaphthalene skeleton in weed control. Furthermore, it claims the use of compounds containing a polyhydronaphthalene skeleton in the control of amaranth or goosegrass.

[0022] The beneficial effects of this invention are:

[0023] The compound described in this invention (attached) Figure 1 It was prepared from the crude fermentation extract of the tobacco endophytic fungus *Aspergillus japonicus* TE-739D. The results were obtained by high-resolution mass spectrometry (HRESIMS). Figure 2 ) and superconducting nuclear magnetic resonance (NMR) spectroscopy (with Figure 3 , 4 A detailed structural identification of the compound was performed, revealing that it contains a polyhydronaphthalene skeleton. (The text then repeats the information using SciFinder.) nDatabase searches revealed that this compound has a novel structure, and it has been named japonicidiene B. The preparation method provided by this invention enables the efficient isolation and preparation of japonicidiene B. This novel compound, japonicidiene B, exhibits significant herbicidal activity against Amaranthus retroflexus and Eleusine indica. Attached Figure Description

[0024] 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.

[0025] Figure 1 This is a molecular structure diagram of the compound japonidiene B prepared in Example 1 of the present invention.

[0026] Figure 2 This is a high-resolution mass spectrum of the compound japonicine B prepared in Example 1 of the present invention.

[0027] Figure 3 The photon spectrum (600 MHz, DMSO-d6) of the compound japoniciene B prepared in Example 1 of this invention is shown.

[0028] Figure 4 The carbon spectrum (150MHz, DMSO-d6) of the compound japoniciene B prepared in Example 1 of this invention is shown. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0030] Example 1

[0031] A method for preparing a compound containing a polyhydronaphthalene skeleton (named japonidiene B) is as follows:

[0032] (1) In a sterile operating table, the mycelia of the activated strain Aspergillus japonicus TE-739D were picked and inoculated onto PDA agar plates and cultured in a constant temperature incubator for 5 days at 28℃. After the culture was completed, the mycelial blocks (1cm×1cm) on the PDA plates were cut and transferred to 1L Erlenmeyer flasks (300mL per flask) containing Potato Dextrose Water (brand: Haibo Biotechnology; number: HB0233-4). The culture medium had been sterilized at high temperature (121℃, 20min).

[0033] (2) After inoculation, the Erlenmeyer flask was allowed to ferment at room temperature for 30 days, with light and dark treatments for 12 hours each. After fermentation, the culture medium was extracted with ethyl acetate (300 mL / bottle) using ultrasonic extraction (ultrasonic power 800 W, ultrasonic working time 30 min) and concentrated under vacuum (vacuum degree 0.09 MPa) to obtain crude extract.

[0034] (3) After dissolving the crude extract in methanol, 100-200 mesh silica gel was added and mixed. Then, the components were separated by silica gel column chromatography under reduced pressure (vacuum degree 0.05Mpa). The components were eluted by 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.

[0035] (4) The fraction Fr.5 eluted from the petroleum ether-ethyl acetate mixture of 30:70 and 0:100 was subjected to gradient elution by LobarLiChroprep RP-18 reverse silica gel column chromatography with methanol-water (volume ratios of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) to obtain 6 subfractions Fr.5.1-5.6;

[0036] (5) The subfraction Fr.5.2 (elution system of 40% methanol-water) was first separated by Sephadex LH-20 gel chromatography column and then purified by semi-preparative high performance liquid chromatography (24% acetonitrile-water) to finally obtain the compound japonidiene B.

[0037] Example 2

[0038] In this embodiment, the molecular structure of the compound japonidiene B prepared in Example 1 was determined by high-resolution mass spectrometry and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy. The physicochemical properties of the compound japonidiene B are as follows:

[0039] Properties: Pale yellow oily substance; Solubility: Easily soluble in DMSO and methanol; Molecular formula: C 20 H 30 O6; Specific rotation: [α] 25 D +77.7 (c=0.26, MeOH); UV absorption spectrum λ max 240 nm; High-resolution mass spectrometry (HRESIMS): m / z 367.2114 [M+H] + (Theoretical value C) 20 H 31 O6 + ,367.2115); 1H NMR spectrum ( 1 HNMR data and carbon spectra ( 13 The C NMR data are shown in Table 1.

[0040] Table 1: NMR data (MDSO-d6) of the compound japonidiene B.

[0041]

[0042] Example 3

[0043] This embodiment uses a seed germination inhibition method to determine the inhibitory activity of the compound japonidiene B prepared in Example 1 on the seed germination of Amaranthus retroflexus and Eleusine indica. The results are shown in Table 2. The specific process is as follows:

[0044] (1) Seed preparation process

[0045] 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.

[0046] 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.

[0047] (2) Preparation of test reagents

[0048] Preparation of the stock solution containing the compound: Weigh 0.3 mg of the compound japonidiene B and add it to 1.5 mL of methanol solution to prepare a stock solution with a concentration of 0.2 mg / mL.

[0049] 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.

[0050] (3) Activity test

[0051] In a clean bench, sterilized filter paper was placed into 12-well plates. For the treatment group, 300 μL of stock solution containing the compound japonidiene B was added to each well, and the plates were allowed to stand until the methanol solvent was dried (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. Three replicates were performed, with 10 seeds added to each replicate. After sealing, the plates were incubated in an artificial incubator for 96 h. Root and shoot lengths were measured, and the inhibition rates of japonidiene B and glyphosate on the germination of the two weed seeds were calculated. The calculation formula is as follows:

[0052] Bud (root) inhibition rate (%) = [Bud ​​(root) length of blank control group - Bud (root) length of treated group] / Bud (root) length of blank control group × 100%

[0053] The results showed that, as shown in Table 2, the compound japonicadiene B exhibited certain inhibitory activity against the germination of Amaranthus retroflexus and Eleusine indica seeds. Specifically, at a concentration of 0.2 mg / mL, japonicadiene B inhibited the shoot growth of Amaranthus retroflexus seeds by 55.82%, which was superior to the positive control glyphosate (45.08%); and it inhibited the shoot growth of Eleusine indica seeds by 93.00%, which was also superior to the positive control glyphosate (79.69%).

[0054] Table 2: Inhibitory activity of the compound japonicadiene B on seed germination of Amaranthus retroflexus and Eleusine indica (0.2 mg / mL)

[0055]

[0056]

[0057] The data in Table 2 are the mean ± standard error.

[0058] 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.

[0059] 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 containing a polyhydronaphthalene skeleton, characterized in that, The compound has the molecular formula C. 20 H 30 O6, the molecular structure is as follows:

2. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 1, characterized in that, Select mycelia of Aspergillus japonicus TE-739D for fermentation culture. The fermentation broth is then separated and purified. Aspergillus japonicus TE-739D is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40901, classified as Aspergillus japonicus, on October 25, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

3. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 2, characterized in that, Specifically, the steps include the following: (1) Select Aspergillus japonicus TE-739D mycelium for fermentation culture to obtain fermentation broth; (2) The fermentation broth was extracted with ethyl acetate and concentrated to obtain a crude extract. (3) After mixing the crude extract with silica gel, gradient elution was performed by vacuum silica gel column chromatography. (4) Collect the fraction Fr.5 eluted under the elution conditions of petroleum ether to ethyl acetate volume ratio of (0:10)-(3:7) and elute it through a reversed-phase silica gel column; (5) The subfraction Fr.5.2 eluted under 40% methanol-water conditions was purified by gel column chromatography and semi-preparative high performance liquid chromatography to obtain the compound as described in claim 1.

4. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 3, characterized in that, The specific operation of step (1) is as follows: the mycelium of the activated strain Aspergillus japonicus TE-739D is picked and inoculated onto PDA plate medium, cultured at 28℃ for 5 days, the mycelium block on the PDA plate is cut and transferred to potato glucose water medium, cultured at room temperature for 30 days to obtain fermentation broth.

5. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 3, characterized in that, The specific operation of step (2) is as follows: the fermentation broth in step (1) is extracted with ethyl acetate by ultrasonic extraction and concentrated under vacuum to obtain crude extract. The conditions for vacuum concentration are: vacuum degree 0.09 MPa, and concentration to non-fluid.

6. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 3, characterized in that, The specific operation of step (3) is as follows: after dissolving the crude extract in methanol, add 100-200 mesh silica gel and mix. Then, separate the components by vacuum silica gel column chromatography with a vacuum degree of 0.05 MPa. Use 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 for elution. Use thin-layer chromatography to combine similar components to obtain 7 components Fr.1-Fr.7 with polarity from small to large.

7. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 6, characterized in that, The specific operation of step (4) is as follows: the fraction Fr.5 eluted from the petroleum ether-ethyl acetate 30:70 mixed system and the 0:100 mixed system is eluted by LobarLiChroprep RP-18 reverse silica gel column chromatography gradient elution. The elution system is methanol-water volume ratio of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, and the fractions are separated and combined to obtain 6 subfractions Fr.5.1-5.

6.

8. The method for preparing a compound containing a polyhydronaphthalene skeleton according to claim 7, characterized in that, The subfraction Fr.5.2 obtained by elution system of 40% methanol-water was first separated by Sephadex LH-20 gel chromatography column and then purified by semi-preparative high performance liquid chromatography with mobile phase of 24% acetonitrile-water to obtain the compound as described in claim 1.

9. The application of the compound containing the polyhydronaphthalene skeleton as described in claim 1 in weed control.

10. The application of the compound containing the polyhydronaphthalene skeleton as described in claim 1 in the control of Amaranthus retroflexus or Eleusine indica.

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