A mixed-origin terpenoid compound from a medicinal mangrove fungus and its application in controlling cotton bollworm.

Abstract

This invention relates to a mixed-origin terpenoid compound from medicinal mangrove fungi and its application in controlling cotton bollworm, wherein the mixed-origin terpenoid compound has the structure shown in compound 1:

Description

Technical Field

[0001] This invention belongs to the field of secondary metabolites of mangrove fungi, specifically relating to a mixed-origin terpene compound from medicinal mangrove fungi and its application in controlling cotton bollworm. Background Technology

[0002] Mangrove plants grow in the tropical and subtropical intertidal zone, where the environment is characterized by high pressure, high salinity, and low oxygen. This environment fosters unique metabolic pathways in their endophytic fungi, enabling them to produce compounds with novel structures and diverse biological activities. Their metabolites possess various medicinal values, including antibacterial, antitumor, immunomodulatory, and enzyme-inhibiting properties, making them a potential resource for microbial drug development. Therefore, mangrove endophytic fungi will become an important resource for new drug research. The applicant's previous Chinese invention patent (CN 116287048 A) disclosed the isolation of a series of diphenyl ether compounds from the fermentation product of fungus TGGP35. This invention further investigates fungus TGGP35 and obtains a mixed-origin terpene compound with applications in controlling cotton bollworm. Summary of the Invention

[0003] This invention provides a mixed-origin terpene compound or its pesticide-acceptable salt from medicinal mangrove fungi, characterized in that the mixed-origin terpene compound has the structure shown in compound 1:

[0004]

[0005] This invention provides a method for preparing mixed-source terpenoid compound 1, characterized by comprising the following steps:

[0006] (1) Inoculate the fungal strain TGGP35 into the fermentation medium and culture it at room temperature for 28-30 days to obtain the fermentation product.

[0007] (2) Extract the fermentation product obtained in step (1) with 1-2 times the volume of ethyl acetate 2-4 times, combine the ethyl acetate phases and concentrate under reduced pressure to obtain the extract.

[0008] (3) The extract obtained in step (2) was subjected to vacuum silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient. The eluent obtained from gradient 90:10 was combined and concentrated. Then, it was subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate (15:1-10:1) as the eluent. Five column volumes were eluted. After vacuum concentration, it was prepared by high-performance liquid chromatography (HPLC). The chromatographic column was a Waters C18, 9.4×250mm, 7μm, the flow rate was 2mL / min, and the mobile phase was MeOH:H2O = 20:80. Compound 1 was obtained. The structure of compound 1 is as follows:

[0009]

[0010] The proportions of the eluent or mobile phase are all volume ratios; the fermentation medium is preferably rice solid culture medium, and the preferred formulation is 50g rice, 60g water, 0.5g sea salt, and 1.5g peptone added to a 1L conical flask.

[0011] The present invention provides a pesticide composition, characterized in that any one of the above-mentioned compound 1 or a pharmaceutically acceptable salt thereof is used as the active ingredient.

[0012] The pesticide composition provided by the present invention also contains other active ingredients; it may also contain pesticide-acceptable carriers or excipients.

[0013] Another embodiment of the present invention provides the use of the compound 1 described herein or a pharmaceutically acceptable salt thereof in the control of cotton bollworm.

[0014] In this invention, the term "pharmaceutically acceptable salt" refers to the addition salt of a nontoxic inorganic or organic acid and / or base, see "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201–217.

[0015] The fungus TGGP35 described in this invention was isolated from the medicinal mangrove plant *Acanthus ilicifolius* L., which was collected by the inventors in August 2015 from the Dongzhaigang Mangrove Nature Reserve in the South China Sea, Hainan Province. Fungus TGGP35 was identified as *Talaromyces flavus* (genus *Talaromyces*) based on morphological characteristics and molecular biological methods (ITS-rRNA sequence alignment) using 18S rRNA amplification and ITS sequencing. The sequence of the ITS region of this fungus has been submitted to NCBI (GenBank accession No. MT071116). The "fungus TGGP35" described in this invention has been disclosed in the inventor's previous research paper "Marine Drugs 2022, 20, 361, Talaromarins A–F: Six New Isocoumarins from Mangrove-Derived Fungus Talaromyces flavus TGGP35" and the previous Chinese invention patent (CN116287048 A). This invention cites in its entirety the contents of the Chinese invention patent (CN 116287048 A). Attached Figure Description

[0016] Figure 1 This is a morphological diagram of the TGGP35 strain;

[0017] Figure 2 It is compound 1 1 H- 1 H COSY Related signal diagram of HMBC (H→C);

[0018] Figure 3 This is the ROESY spectrum of compound 1;

[0019] Figure 4 This is the experimental ECD spectrum of compound 1;

[0020] Figure 5 It is compound 1 1 H NMR spectrum;

[0021] Figure 6 It is compound 1 13 C NMR spectrum;

[0022] Figure 7 This is the 135°-DEPT plot of compound 1;

[0023] Figure 8 This is the HMQC diagram of compound 1;

[0024] Figure 9It is compound 1 1 H- 1 H COSY diagram;

[0025] Figure 10 This is the ROESY diagram of compound 1;

[0026] Figure 11 This is the HR-ESI-MS image of compound 1. Detailed Implementation

[0027] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0028] Example 1

[0029] (1) Preparation of fermentation medium: The formula is to add 50g rice, 60g water, 0.5g sea salt and 1.5g peptone to each conical flask (1L conical flask). Incubate at 120℃ for 25–30 minutes.

[0030] The fungal strain TGGP35 was inoculated into fermentation medium (200 bottles) and cultured statically at room temperature for 30 days to obtain the fermentation product.

[0031] (2) The fermentation product obtained in step (1) was extracted three times with an equal volume of ethyl acetate. The ethyl acetate phases were combined and concentrated under reduced pressure to obtain an extract.

[0032] (3) The extract obtained in step (2) was subjected to vacuum silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient. The eluent obtained from gradient 90:10 was concentrated and then subjected to normal phase silica gel column chromatography with a mixed solvent of petroleum ether:ethyl acetate = 15:1-10:1. Five column volumes were eluted, concentrated under reduced pressure, and then prepared by high performance liquid chromatography (HPLC). The chromatographic column was a Waters C18, 9.4×250mm, 7μm, the flow rate was 2mL / min, and the mobile phase was MeOH:H2O = 20:80, yielding compound 1 (4.2mg). The structure of compound 1 is as follows:

[0033]

[0034] Compound 1: White powder. High-resolution mass spectrometry (HR-ESI-MS) showed a quasi-molecular ion peak [MH] at m / z 455.2081. -The molecular formula of the compound was deduced to be C. 26 H 32 O7 has an unsaturation degree of 11. 1 The H-NMR spectrum shows that this compound contains two olefin hydrogen signals [δ]. H [6.65 (d, J = 9.6 Hz, H⁻¹) and 6.11 (d, J = 9.6 Hz, H⁻²)], two methylene hydrogen signals appear in the oxygen-bound region [δ]. H 5.42 (s, H-11) and 4.23 (q, J = 6.4 Hz, H-5')] and 1 methyl hydrogen signal δ H 3.57 (s, 11-OMe), two methylene hydrogen signals appear in the high-field region [δ] H 1.68 (m, H-7) and δ H 1.58 (m, H-6)] and 7 methyl hydrogen signals [δ H 2.15(s,H-7'), 1.84(s,H-8'), 1.62(s,H-13), 1.52(s,H-9'), 1.40(s,H-15), 1.33(s,H-14) and 1.10(s,H-12)]; combined with 13 The C-NMR spectrum revealed 26 resonance carbon signals in this compound, including two ester carbonyl carbon signals (δ). C (172.4, 164.3), 6 olefin carbon signals δ C (160.2, 146.5, 138.4, 138.4, 136.7, 120.3), 5 seasonal carbon signals δ C (85.7, 84.4, 62.8, 45.6, 43.2), two δ-hydroxymethyl carbon signals C (99.2, 75.0), 1 methoxy carbon signal δ C 56.3, 2 methylene carbon signals δ C (26.0, 25.6), 7 methyl carbon signals δ C (25.8, 23.2, 23.1, 15.8, 15.5, 14.2, 8.8). Combining the above 1D NMR data, this compound exhibits a similar mixed-origin terpene skeleton to brasilianoid G in the literature. The most significant difference lies in the addition of a methoxy group and a methyl group. Furthermore, the HMBC spectrum shows a correlation between H-11 and 11-OMe / C-9, and between H-13(3H) and C-5 / 9 / 10. These data indicate the attachment of an oxymethyl group (δ-hydroxymethyl group) at C-11. C 56.3) Replace the aldehyde group (δ) on the original C-11 of brasilianoid G. C199.9), and at position C-13, the original double bond (δ) of brasilianoid G. C 128.8) was replaced with a methyl group (δ) C 15.8). Furthermore, analysis of the ROESY spectrum reveals that H-1 is correlated with 11-OMe(H) / H-9', H-11 with 11-OMe(H), and H-12 with H-14. Thus, the planar structure of compound 1 is determined. The absolute configuration of this compound was directly determined by ECD calculations, exhibiting a negative Cotton effect (-11.89 nm) at 264 nm. Therefore, the absolute configuration of this compound is determined to be 5R,8S,11S,1'S,5'R,6'R,11R'.

[0035] Nuclear magnetic resonance (NMR) data of compound 1 (CDCl3)

[0036]

[0037]

[0038] Example 2 Insect resistance activity test:

[0039] 1. Experimental instruments and materials: constant temperature incubator, pipette, electronic balance, brush, feed, azadirachtin, dimethyl sulfoxide (DMSO) and cotton bollworm larvae.

[0040] 2. Using an electronic balance, evenly distribute 6g of feed into a 6-well plate. The concentration gradient of the test compound 1 is 200, 100, and 50 μg / mL. DMSO is used as a negative control, azadirachtin as a positive control, and artificial feed as a blank control. Mix the test sample and feed thoroughly. Place two cotton bollworm larvae of the same size into each well of the 6-well plate. Each test is repeated three times. Finally, place the plate in a constant temperature incubator and observe for 1-2 weeks. Observe and record the growth status of the cotton bollworms every 2 days to determine whether the larvae have died or their growth has been inhibited.

[0041] 3. Experimental Results:

[0042]

Claims

1. A mixed-origin terpene compound or its pesticide-acceptable salt from a medicinal mangrove fungus, characterized in that... The mixed-source terpenoid compound has the structure shown in compound 1:

2. A method for preparing mixed-source terpenoid compound 1, characterized in that... Includes the following steps: (1) Inoculate the fungal strain TGGP35 into the fermentation medium and culture it at room temperature for 28-30 days to obtain the fermentation product. (2) Extract the fermentation product obtained in step (1) with 1-2 times the volume of ethyl acetate 2-4 times, combine the ethyl acetate phases and concentrate under reduced pressure to obtain the extract. (3) The extract obtained in step (2) was subjected to vacuum silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate as the eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:

100. Two column volumes were collected for each gradient. The eluent obtained from gradient 90:10 was combined and concentrated. Then, it was subjected to normal-phase silica gel column chromatography with a mixed solvent of petroleum ether and ethyl acetate (15:1-10:1) as the eluent. Five column volumes were eluted. After vacuum concentration, it was prepared by high-performance liquid chromatography (HPLC). The chromatographic column was a Waters C18, 9.4×250mm, 7μm, the flow rate was 2mL / min, and the mobile phase was MeOH:H2O = 20:

80. Compound 1 was obtained. The structure of compound 1 is as follows:

3. The method according to claim 2, characterized in that... The fermentation medium is selected from rice solid culture medium, and the formula is 50g rice, 60g water, 0.5g sea salt and 1.5g peptone added to a 1L conical flask.

4. A pesticide composition, characterized in that... The active ingredient is compound 1 as described in claim 1 or a pesticide-acceptable salt thereof.

5. The pesticide composition according to claim 4, characterized in that... The pesticide composition also contains other active ingredients.

6. The pesticide composition according to any one of claims 4-5, characterized in that... The pesticide composition also contains a pesticide-acceptable carrier or excipient.

7. The use of compound 1 of claim 1 or a pesticide-acceptable salt thereof in the control of cotton bollworm.

Images