A marine fungal anti-inflammatory compound, its preparation method and application

Abstract

This invention belongs to the field of marine natural product chemistry, specifically relating to an anti-inflammatory compound from a marine fungus, its preparation method, and its application. This invention utilizes a combination of chromatographic separation and spectroscopic identification techniques to obtain 13 mixed-origin terpenoid compounds from the marine fungus 23061902, among which compound 1 is a novel compound. Anti-inflammatory activity tests showed that compound 8 significantly inhibited LPS-induced NO release in RAW264.7 cells, with an IC50 value of [missing information]. 50 The concentration was 60.01 µM. Compound 8 inhibited the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-10, and IL-6 in LPS-induced RAW264.7 cells in a dose-dependent manner. This invention provides a material basis and theoretical foundation for the development of anti-inflammatory drugs, while also taking into account the sustainable development and utilization of medicinal mangrove resources.

Description

Technical Field

[0001] This invention belongs to the field of marine natural product chemistry, specifically relating to an anti-inflammatory compound of marine fungi, its preparation method, and its application. Background Technology

[0002] Mangrove-derived fungi, due to the unique habitats of their hosts—high salinity, strong light, and acidity—produce novel, diverse, and bioactive secondary metabolites. Currently, over 1500 new compounds have been isolated from mangrove-derived fungi, exhibiting various biological activities such as antibacterial, antitumor, and anti-inflammatory effects, making them an important resource for natural product drug development. Compounds of various structural types, including terpenoids, polyketides, alkaloids, and peptides, have been discovered from mangrove-derived *Talaromyces* sp. fungi, showing anti-inflammatory, antioxidant, antitumor, and antibacterial activities. For example, talaroacids A, B, and D significantly inhibit LPS-induced NO release in RAW264.7 cells; talatensindoids A and B exhibit strong inhibitory activity against various plant pathogenic fungi; and talarotone C has significant antitumor activity. These existing technologies demonstrate that mangrove-derived *Talaromyces* sp. fungi possess significant development potential and application value. This invention discovered that the crude extract of *Talaromyces* sp. 23061902 has a certain inhibitory effect on LPS-induced NO production in RAW264.7 cells, and obtained 13 mixed-origin terpenoid compounds 1-13 from it. Among them, compound 1 is a new compound, and compound 8 has the strongest inhibitory effect on LPS-induced NO production in RAW264.7 cells (IC50). 50 The value was 60.01 µM, and its effect on the expression of pro-inflammatory factors TNF-α, IL-1β, IL-10 and IL-6 was evaluated. Summary of the Invention

[0003] This invention provides a mangrove stem fungus, *Talaromyces sp.* 23061902 (hereinafter referred to as marine fungus 23061902), characterized by its preservation information: Preservation Institution: Guangdong Provincial Center for Microbial Culture Collection; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology; Preservation Date: April 25, 2025; Accession Number: GDMCC No. 66210; Classification: *Talaromyces sp.*. This marine fungus was isolated from the stem of *Talaromyces simonii*, a mangrove plant, in the Bamen Bay Mangrove Nature Reserve, Wenchang City, Hainan Province.

[0004] Another embodiment of the present invention provides an anti-inflammatory active compound or a pharmaceutically acceptable salt thereof, characterized in that the anti-inflammatory active compound has the structure shown in compound 1:

[0005] .

[0006] Another embodiment of the present invention provides an ethyl acetate extract of the above-mentioned marine fungus 23061902, characterized in that the preparation method of the ethyl acetate extract includes the following steps:

[0007] (1) Marine fungus 23061902 was inoculated into rice culture medium and fermented at room temperature for 30 days to obtain fermentation product;

[0008] (2) The fermentation product obtained in step (1) is soaked in an equal volume of ethyl acetate for extraction 2-4 times, with an interval of 4-6 days between each extraction. The extracts are combined and concentrated under reduced pressure to obtain the ethyl acetate extract.

[0009] In step (1), the rice culture medium is formulated to contain 50 g of rice and 50 mL of artificial seawater in each 1 L Erlenmeyer flask.

[0010] Another embodiment of the present invention provides the use of the above-mentioned marine fungus 23061902 in the preparation of any one or more of compounds 1-13 or in the above-mentioned ethyl acetate extract; the structures of compounds 1-13 are as follows:

[0011]

[0012] Another embodiment of the present invention provides the use of any one or more of the above-described compounds 1-13 or their pharmaceutically acceptable salts, or the above-described ethyl acetate extracts, in the preparation of anti-inflammatory drugs.

[0013] Another embodiment of the present invention provides an anti-inflammatory pharmaceutical composition, characterized in that the pharmaceutical composition uses one or more of compounds 1-13 or their pharmaceutically acceptable salts, or the above-described ethyl acetate extracts, as active ingredients. The pharmaceutical composition may also optionally contain pharmaceutically acceptable excipients. The pharmaceutical composition may also optionally contain other anti-inflammatory active ingredients.

[0014] Compared with the prior art, this invention discloses a marine fungus 23061902 derived from the stem of *Hymenochloa chinensis*, and studies its secondary metabolites. Using a combination of chromatographic separation techniques such as normal and reversed-phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative HPLC, along with modern spectroscopic identification techniques such as MS, NMR, computational NMR, and ECD, 13 mixed-source terpenoid compounds were isolated and identified from the ethyl acetate extract of marine fungus 23061902. These compounds were identified as dhilirolides O(1), D(2), J(3), A(4), B(5), L(6), talarine A(7), talaromyolides C(8), E(9), K(10), talarine H(11), berkeleytrion(12), and berkeleyone C(13). Among these, compound 1 is a new compound, and its relative and absolute configurations were determined using NOESY spectra, DP4+, and ECD calculations. Activity screening revealed that compound 8 had a certain inhibitory effect on lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells, with an IC50 value of [missing value]. 50 The concentration was 60.01 µM. Compound 8 inhibited the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-10, and IL-6 in LPS-induced RAW264.7 cells in a dose-dependent downregulation manner, thereby exerting its anti-inflammatory effect. The ethyl acetate extract and other compounds also showed varying degrees of inhibition on NO production in LPS-induced RAW264.7 cells. Attached Figure Description

[0015] Figure 1 It is the key to compound 1 1 H- 1 H COSY and HMBC related signal diagram.

[0016] Figure 2 The key to compound 1 is NOESY ( Related signal diagram.

[0017] Figure 3 These are the test CD and calculated ECD spectra of compound 1.

[0018] Figure 4 This is a graph showing the inhibitory activity of compound 8 on NO in LPS-treated RAW264.7 cells, n = 3, P < 0.001. C: blank control, DEX: dexamethasone.

[0019] Figure 5This is a graph showing the effect of compound 8 on the viability of RAW264.7 cells, n = 3, P < 0.001. Con: blank control.

[0020] Figure 6 The figure shows the effect of compound 8 on the levels of inflammatory factors in LPS-treated RAW264.7 cells. The levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 10 (IL-10), and interleukin 6 (IL-6) were measured by ELISA, n = 3, P < 0.001. Detailed Implementation

[0021] 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.

[0022] Example 1

[0023] 1. Instruments and Materials

[0024] Bruker AV-400 NMR spectrometer (Bruker GmbH, Germany); Thermo Fisher Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, USA); Bruker Esquire HCT mass spectrometer (Bruker GmbH, Germany); JASCO P-1020 polarimeter (Electronics Corporation, Japan); JASCO J-715 circular dichroism spectrometer (JASCO Corporation, Japan); Agilent 1200 semi-preparative high-performance liquid chromatograph (Agilent Technologies, USA); Eclipse XDB-C18 semi-preparative column (9.4 mm × 250 mm, 5 μm); column chromatography silica gel (100–200, 200–300, 300–400 mesh) and thin-layer chromatography silica gel (Qingdao Marine Chemical Plant); Sephadex LH-20 (MedChemExpress, USA). LLC); reversed-phase silica gel packing material (ODS, 10-50 μm particle size, Merck, Germany); all reagents, such as chromatographic grade acetonitrile, analytical grade methanol, analytical grade ethyl acetate, and analytical grade petroleum ether, etc. (Guangdong Xilong Chemical Co., Ltd.).

[0025] 2. Source of strain

[0026] The medicinal mangrove plant *Talaromyces sp.* was collected in September 2022 from the Bamen Bay Mangrove Nature Reserve in Wenchang City, Hainan Province. A fungus, named 23061902, was isolated, identified, and screened from the stem of *Talaromyces sp.*. Based on morphological observation and molecular biological methods, the fungus was identified as *Talaromyces sp.*. The preservation information for this strain is as follows: Depository Institution: Guangdong Provincial Center for Microbial Culture Collection; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology; Deposit Date: April 25, 2025; Accession Number: GDMCC No. 66210; Classification: *Talaromyces sp.*. It is also deposited at the Key Laboratory of Tropical Medicinal Resources Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University.

[0027] 3. Preparation of ethyl acetate extract and isolation of compounds 1-13

[0028] Pretreatment and activation of the seed culture is a routine procedure in this field. For example, the following method is used: Marine fungus 23061902 is taken out of the -80 ℃ ultra-low temperature freezer for activation, inoculated onto potato dextrose agar (PDA) medium, and placed in a constant temperature incubator at 27 ℃ for 2-3 days to observe its growth. Then, the strain is picked with a sterilized inoculation needle and inoculated into Erlenmeyer flasks containing potato dextrose water (PDB) medium. Each 1 L Erlenmeyer flask contains 300 mL of PDB medium. The flasks are shaken at 28 ℃ and 120 r / min for 5 days to observe the growth of the seed culture. The seed culture is obtained when the seed culture is evenly distributed in the medium by shaking.

[0029] (1) Inoculate marine fungus 23061902 (seed liquid) into rice culture medium (each 1L conical flask contains 50 g rice and 50 mL artificial seawater), inoculate 50 flasks, and let them ferment at room temperature for 30 days to obtain fermentation product;

[0030] (2) The fermentation product obtained in step (1) was soaked in ethyl acetate of equal volume three times, with an interval of 5 days between each extraction. The extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract (about 25g).

[0031] (3) Isolation of compounds 1-13

[0032] The ethyl acetate extract obtained in step (2) was eluted by normal phase silica gel column chromatography (100 ~ 200 mesh) gradient and separated into 8 fractions (Fr.1 ~ Fr.8) by petroleum ether / ethyl acetate [V (petroleum ether) / V (ethyl acetate), gradient 100:0 → 0:100] and ethyl acetate / methanol [V (ethyl acetate)V (methanol), gradient 100:0 → 0:100]. Fr.8 (1.2 g) was purified by normal-phase silica gel column chromatography using a gradient elution of petroleum ether / ethyl acetate [V(petroleum ether) / V(ethyl acetate), 9:1 → 1:1], and the fractions were combined into 6 fractions (Fr.8.1 ~ Fr.8.6) after TLC analysis; Fr.8.4 (0.5 g) was purified by semi-preparative HPLC separation using a Sephadex LH-20 gel column [V(petroleum ether) / V(chloroform) / V(methanol) = 2:1:1] to obtain compounds 1 (5.5 mg), 2 (3.6 mg), 3 (6.5 mg), 4 (4.8 mg), 5 (4.6 mg) and 6 (4.5 mg). Fr.6 (1.8 g) was eluted using a Sephadex LH-20 gel column to obtain five fractions (Fr.6.1~Fr.6.5); Fr.6.3 (1.0 g) was eluted using a normal-phase silica gel column [V(petroleum ether) / V(ethyl acetate), 10:1 → 1:1] gradient, and then purified by semi-preparative HPLC to obtain compounds 7 (6.0 mg), 8 (8.0 mg), and 9 (7.6 mg). Fraction Fr.5 (1.1 g) was eluted using a normal-phase silica gel column with a petroleum ether / ethyl acetate gradient [V(petroleum ether) / V(ethyl acetate), 5:1 → 0:1] to obtain three fractions (Fr.5.1 ~ Fr.5.3); Fr.5.2 (0.4 g) was purified by Sephadex LH-20 gel column chromatography and semi-preparative HPLC to obtain compounds 10 (4.4 mg) and 11 (6.5 mg). Fraction Fr.4 (1.1 g) was eluted with a normal-phase silica gel column using a gradient of petroleum ether / ethyl acetate [V(petroleum ether) / V(ethyl acetate), 8:1→0:1] to obtain three fractions (Fr.4.1 ~ Fr.4.3); Fr.4.2 (0.2 g) was purified by Sephadex LH-20 gel column and semi-preparative HPLC to obtain compound 12 (4.4 mg); Fr.4.3 (0.1 g) was purified by Sephadex LH-20 gel column and semi-preparative HPLC to obtain compound 13 (3.5 mg). Note: Unless otherwise specified, the eluent for the Sephadex LH-20 gel column was [V(chloroform) / V(methanol) = 1:1], and the mobile phase for the semi-preparative HPLC was [V(water) / V(methanol) = 50:50→20:80].

[0033]

[0034] 4. Structural identification of compounds 1-13

[0035] Compound 1 is a yellow powder, with an HR-ESI-MS m / z of 463.2 [M+Na]. + , combined 1 H NMR and 13 CNMR data (Table 1) suggest the molecular formula is C1. 25 H 26 O7 has an unsaturation degree of 13. 1 The H NMR spectrum shows two olefinic proton signals δ in the low-field region. H 6.04 (s, H-5) and 5.97 (s, H-2), δ of the terminal olefin proton signal H 5.23 (dd, J = 16.8, 0.8 Hz, H-22), δ of a group of hydroxymethylene proton signals H 2.73 (d, J = 5.2 Hz, H-15α) and 2.54 (d, J = 5.2 Hz, H-15β), a single hydroxymethyl proton signal δ H 4.48 (d, J = 7.2Hz, H-23), 2 sets of methylene signals [(δ H [3.38 (dd, J = 18.8, 3.2 Hz, H-6α), 2.73 (dd, J = 18.8, 6.8 Hz, H-6β)] and [1.77 (br t , J = 13.8 Hz, H-12α), 1.60 (dd, J = 13.8, 5.6 Hz, H-12β)], 1 methylene hydrogen signal δ H 2.94 (dd, J = 13.6, 5.6 Hz, H-13), 5 groups of methyl signals δ H 1.55 (s, H-18), 1.45 (s, H-17), 1.35 (s, H-19), 1.24 (s, H-25), 1.15 (d, J = 7.2 Hz, H-24). 13 The CNMR spectrum yielded 25 carbon signals. Based on the DEPT-135 spectrum, it is inferred that this compound contains 3 carbonyl carbon signals (δ). C 215.0 (C-10), 172.2 (C-20), 164.4 (C-1), 6 aromatic carbon signals [δ] C154.8 (C-3), 146.7 (C-21), 133.5 (C-4), 130.6 (C-5), 115.3 (C-2), 109.6 (C-22)], 3 methylene carbon signals δ C 53.9 (C-15), 41.0 (C-12), 38.5 (C-6), 2 methylene carbon signals δ C 84.5 (C-23), 41.3 (C-13), 6 seasonal carbon signals δ C 90.3 (C-9), 83.4 (C-16), 65.6 (C-8), 59.0 (C-14), 55.1 (C-11), 43.3 (C-7), δ of 5 methyl carbons C 26.9 (C-17), 26.5 (C-18), 18.1 (C-24), 17.0 (C-19), 15.4 (C-25). Combining the above... 1 H NMR spectrum and 13 Based on the C10 NMR spectrum information, it is inferred that this compound is a mixed terpene compound, and is similar to dhilirolide D. The difference is that compound 1 lacks a methyl signal, but has an additional set of methylene signals [δ]. C (53.9), δ H 2.73 (d, J = 5.2 Hz) and 2.54 (d, J = 5.2 Hz). Based on HMBC spectral information, H-13 is correlated with CH2-15 and C-14. Combining this with mass spectrometry data, it is inferred that C-14 and C-15 form an oxygen-bonded three-membered ring. Further analysis using COSY and HMBC confirms the planar structure of compound 1. Figure 1 The relative configurations of some chiral carbons in compound 1 were determined by analyzing the NOESY spectrum. 19-CH3 was correlated with 24-CH3 / 25-CH3 / 17-CH3, and 17-CH3 was correlated with H-15β, indicating that 19-CH3 / 24-CH3 / 25-CH3 / H-15β were on the same side. 18-CH3 was correlated with H-13 / H-15α, indicating that H-13 / H-15α were on the other side of the molecule. Figure 2 Since quaternary carbons C-8 and C-9 do not have direct NOESY correlation signals, their relative configurations cannot be determined through NOESY spectra. Further investigation using density functional theory (DFT) is necessary. 13The compounds were identified by C NMR calculations and DP4+ statistical analysis. Four possible relative configurations were determined: (7R*, 8S*, 9S*, 11R*, 13R*, 14R*, 21S*)-1 (1a), (7R*, 8S*, 9R*, 11R*, 13R*, 14R*, 21S*)-1 (1b), (7R*, 8R*, 9S*, 11R*, 13R*, 14R*, 21S*)-1 (1c), and (7R*, 8R*, 9R*, 11R*, 13R*, 14R*, 21S*)-1 (1d). DP4+ statistical analysis was used to compare the experimental and calculated configurations. 13 The 1C NMR data showed that the confidence level of (7R*, 8S*, 9R*, 11R*, 13R*, 14R*, 21S*)-1 (1b) was 100%. Subsequently, the ECD spectra of the (7R, 8S, 9R, 11R, 13R, 14R, 21S)-1 and (7S, 8R, 9S, 11S, 13S, 14S, 21R)-1 configurations were calculated and compared with experimental values. It was found that the theoretical ECD spectra of the (7R, 8S, 9R, 11R, 13R, 14R, 21S)-1 configuration showed a consistent trend with the tested ECD spectra. Figure 3 Thus, the absolute configuration of compound 1 was determined to be 7R,8S,9R,11R,13R,14R,21S, and it was named dhilirolide O.

[0036] Dhilirolide O (1): [α] 20 D -80.0 (c 0.10, MeOH); UV (MeOH) λ max (log ε)220, 270; IR (KBr) ν max 3460, 2964, 2924, 1680, 1625, 1595, 1580 cm -1 . ECD (2.98 mM, CH3OH) λmax (Δε) 229 (+16.19), 254 (+12.87), 273 (-20.22), 299 (+91.91).

[0037] Table 1 Compound 1 1 H NMR and 13 C10 NMR data (CDCl3)

[0038]

[0039] Compound 2 is a yellow powdery solid with the molecular formula C. 25 H 30 O7. 1 H NMR (CD3OD, 400 MHz) δ H : 6.22 (1H, s, H-2), 6.11 (1H, m, H-5), 5.19 (1H, s, H-22), 5.05 (1H, s, H-22), 4.46 (1H, q, J = 7.2Hz, H-23), 3.59, (1H, dd, J = 20.0, 3.4 Hz, H-6), 2.75 (1H, dd, J = 13.4, 5.4 Hz, H-13), 2.57, (1H, dd, J = 20.0, 6.2 Hz, H-6), 2.04 (1H, bt, J = 13.2 Hz, H-12), 1.97 (1H, dd, J = 13.0, 5.4 Hz, H-12), 1.61 (3H, s, H-18), 1.48 (3H, s, H-17), 1.29 (3H, s, H-15), 1.23 (3H, s, H-25), 1.21 (3H, s, H-19), 1.12 (3H, d, J = 7.0 Hz, H-24). 13 C NMR (CD3OD, 100 MHz) δ C :215.6 (C-10), 174.7 (C-20), 167.6 (C-1), 165.7 (C-3), 150.0 (C-21), 133.2 (C-4) , 132.7 (C-5), 114.9 (C-2), 108.4 (C-22), 91.1 (C-9), 91.1 (C-23), 85.0 (C-16), 76 0.3 (C-14), 67.5 (C-8), 56.7 (C-11), 50.8 (C-13), 44.4 (C-7), 43.2 (C-12), 40.1 (C-6), 33.5 (C-15), 27.2 (C-17), 26.8 (C-18), 18.5 (C-24), 16.7 (C-19), 16.0 (C-25). Combining the above... 1 H NMR spectrum and 13 Based on the C NMR spectral information, it was inferred that the compound was a mixed-origin terpene. By comparing the data with those in the literature (J Org Chem, 2014, 79(8): 3327.), the compound was identified as dhilirolide D.

[0040] Compound 3 is a yellow powdery solid with the molecular formula C3. 25 H 28 O7. ¹H NMR (CDCl3, 400 MHz) δH : 6.06 (1H, m, H-5), 5.78 (1H, s, H-2), 5.17 (1H, s, H-22), 5.12 (1H, s, H-22), 4.72 (1H, q, J = 7.0Hz, H-23), 3.11 (1H, d, J = 19.4 Hz, H-6), 2.15 (1H, dd, J = 19.6, 5.6 Hz, H-6), 2.07 (1H, d, J = 12.2 Hz, H-12), 1.61 (3H, s, H-18), 1.55 (3H, s, H-17), 1.51 (3H, s, H-15), 1.48 (1H, d, J = 12.2 Hz, H-12), 1.23 (3H, d, J = 6.0 Hz, H-24), 1.23 (3H, s, H-25), 1.06 (3H, s, H-19). ¹³C NMR (CDCl3, 100 MHz) δ C :171.8 (C-20), 164.0 (C-1), 150.8 (C-3), 148.8 (C-21), 134.2 (C-4), 130.4 (C-5), 115.2 (C-2), 109.1 (C-10), 108.6 (C-14), 108.2 (C-22), 92.9 (C-9), 82.6 (C-16), 8 1.4 (C-23), 60.1 (C-8), 49.5 (C-11), 48.2 (C-13), 40.6 (C-7), 39.0 (C-12), 35.5 (C-6), 30.1 (C-17), 28.5 (C-18), 20.8 (C-15), 20.1 (C-19), 18.8 (C-24), 15.7 (C-25). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (JOrg Chem, 2014, 79(8): 3327.), the compound is identified as dhilirolide J.

[0041] Compound 4 is a yellow powdery solid with the molecular formula C4. 25 H 28 O9. ¹H NMR (CDCl3, 400 MHz) δ H: 6.32 (1H, s, H-2), 4.77 (1H, m, H-23), 3.33 (1H, d, J = 6.0 Hz, H-5), 2.84 (1H, d, J = 4.0 Hz, H-22), 2.67 (1H, bd, J = 14.8 Hz, H-6), 2.58 (1H, d, J = 4.0 Hz, H-22), 2.02 (1H, d, J = 12.8 Hz, H-12), 1.95 (1H, dd, J = 14.8, 6.4 Hz, H-6), 1.89 (1H, d, J = 12.8 Hz, H-12), 1.63 (3H, s, H-15), 1.56 (3H, s, H-18), 1.53 (3H, d, J = 7.0 Hz, H-24), 1.40 (3H, d, J = 1.4 Hz, H-19), 1.30 (3H, s, H-17), 0.91 (3H, s, H-25). ¹³C NMR (CDCl3, 100 MHz) δ C : 170.5 (C-20), 162.8 (C-1), 152.7 (C-3), 125.2 (C-2), 109.6 (C-14), 106.3 (C-10 ), 91.9 (C-9), 81.9 (C-16), 81.3 (C-23), 64.2 (C-21), 56.5 (C-5), 56.0 (C-4), 54. 8 (C-8), 48.7 (C-13), 46.1 (C-22), 45.6 (C-11), 41.1 (C-7), 37.0 (C-12), 32.3 (C-6), 27.3 (C-18), 21.8 (C-17), 21.6 (C-15), 21.5 (C-19), 18.6 (C-24), 13.2 (C-25). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (J Org Chem, 2014, 79(8): 3327.), the compound is identified as dhilirolide A.

[0042] Compound 5 is a yellow powdery solid with the molecular formula C. 25 H 28 O8. ¹H NMR (DMSO-d6, 400 MHz) δ H: 7.51 (1H, s, 10-OH), 6.16 (1H, m, H-5), 5.83 (1H, s, H-2), 4.72 (1H, q, J = 7.0 Hz, H-23), 2.93 (2H, m, H-6, H-22), 2.41 (1H, d, J = 3.8 Hz, H-22), 1.98 (1H, d, J = 13.2 Hz, H-12), 1.95 (1H, dd, J = 19.6, 5.6 Hz, H-6), 1.69 (1H, d, J = 13.2 Hz, H-12), 1.55 (3H, s, H-18), 1.51 (3H, s, H-17), 1.49 (3H, s, H-15), 1.44 (3H, d, J = 7.0 Hz, H-24), 1.16 (3H, s, H-19), 0.77 (3H, s, H-25). ¹³C NMR (DMSO-d6, 100 MHz) δ C :170.3 (C-20), 162.8 (C-1), 150.1 (C-3), 133.4 (C-4), 129.4 (C-5), 114.7 (C-2), 107.4 (C-14), 106.4 (C-10), 91.1 (C-9), 82.1 (C-16), 80.8 (C-23), 64.1 (C-21), 54 0.3 (C-8), 47.5 (C-13), 45.9 (C-22), 45.1 (C-11), 40.4 (C-7), 35.4 (C-6), 35.0 (C-12), 29.4 (C-17), 27.8 (C-18), 20.4 (C-19), 20.3 (C-15), 18.2 (C-24), 13.3 (C-25). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (J Org Chem, 2014, 79(8): 3327.), the compound is identified as dhilirolide B.

[0043] Compound 6 is a yellow powdery solid with the molecular formula C6. 25 H 28 O7. ¹H NMR (CDCl3, 400 MHz) δ H: 5.23 (1H, bs, H-22), 5.20 (1H, bs, H-22), 4.66 (1H, bs, H-5), 4.44 (1H, q, J = 7.6 Hz, H-23), 2.95 (1H, d, J = 19.6 Hz, H-2), 2.75 (1H, d, J = 14.2 Hz, H-12), 2.29 (1H, d, J = 19.6 Hz, H-2), 2.19 (1H, dd, J = 14.4, 3.4 Hz, H-6), 2.05 (3H, s, H-15), 1.77 (1H, bd, J = 13.6 Hz, H-6), 1.73 (1H, d, J = 14.4 Hz, H-12), 1.65 (3H, s, H-17), 1.59 (3H, s, H-18), 1.40 (3H, s, H-19), 1.13 (3H, s, H-25), 1.12 (3H, d, J = 7.4 Hz, H-24). ¹³C NMR (CDCl3, 100 MHz) δ C :212.8 (C-10), 203.9 (C-14), 171.4 (C-20), 168.1 (C-1), 147.2 (C-21), 137.4 (C- 4), 129.3 (C-3), 107.9 (C-22), 87.6 (C-9), 84.5 (C-16), 81.6 (C-23), 65.6 (C-5), 6 1.0 (C-8), 59.8 (C-13), 54.2 (C-11), 45.1 (C-12), 37.8 (C-7), 33.9 (C-6), 31.8 (C-2), 28.3 (C-15), 27.1 (C-18), 26.3 (C-17), 18.1 (C-19), 16.9 (C-24), 16.0 (C-25). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (J Org Chem, 2014, 79(8): 3327.), the compound is identified as dhilirolide L.

[0044] Compound 7 is a yellow needle-like crystal with the molecular formula C. 25 H 32 O6. ¹H NMR (CDCl3, 400 MHz) δ H: 11.12 (1H, s, 4'-OH), 6.33 (1H, s, H-5'), 4.62 (1H, m, H-8'), 2.87 (1H, dd, J = 16.6, 3.2 Hz, H-7'), 2.72 (1H, d, J = 16.2 Hz, H-11), 2.58 (1H, m, H-7'), 2.54 (1H, m, H-2), 2.51 (1H, m, H-11), 2.49 (1H, m, H-5), 2.09 (1H, dt, J = 13.2, 4.0 Hz, H-7), 1.86 (1H, dt, J = 13.2, 6.4Hz, H-7), 1.68 (1H, m, H-6), 1.66 (1H, m, H-1), 1.53 (3H, d, J = 6.2 Hz, H-9'), 1.52 (1H, m, H-6), 1.35 (3H, s, H-12), 1.14 (3H, s, H-14), 1.13 (3H, s, H-15), 1.07 (3H, s, H-13). ¹³C NMR (CDCl3, 100 MHz) δ C :217.2 (C-3), 170.0 (C-10'), 162.4 (C-4'), 158.8 (C-6'), 140.4 (C-2'), 109.9 (C- 3'), 104.3 (C-5'), 103.3 (C-1'), 80.5 (C-8), 74.8 (C-8'), 74.4 (C-9), 47.1 (C-4), 45.2 (C-5), 40.9 (C-10), 34.0 (C-7), 33.9 (C-2), 31.5 (C-1), 31.2 (C-7'), 27.4 (C-11), 27.2 (C-14), 23.5 (C-12), 21.2 (C-13), 21.0 (C-9'), 20.3 (C-6), 18.3 (C-15). Based on the above ¹H NMR and ¹³C NMR spectra, this compound is presumed to be a mixed-origin terpene. By comparing with data from the literature (Phytochemistry, 2025, 236, 114493), this compound is identified as talarine A.

[0045] Compound 8 is a yellow powdery solid with the molecular formula C. 26 H 34 O7. ¹H NMR (CDCl3, 400 MHz) δ H: 6.35 (1H, s, H-5), 4.66 (1H, m, H-9), 2.90 (1H, dd, J = 16.4, 3.2 Hz, H-10), 2.70 (1H, d, J = 16.0Hz, H-15), 2.62 (1H, dd, J = 16.4, 11.2 Hz, H-10), 2.55 (1H, d, J = 15.8 Hz, H-15), 2.47 (1H, m, H-22), 2.03 (1H, m, H-27), 1.97 (1H, m, H-26), 1.93 (1H, m, H-21), 1.82 (1 H, m, H-27), 1.73 (1H, m, H-30), 1.69 (2H, m, H-22, H-26), 1.68 (2H, m, H-21, H-26), 1.54 (3H, d, J = 6.2Hz, H-12), 1.29 (3H, s, H-23), 1.12 (3H, s, H-28), 0.88 (3H, d, J = 9.6 Hz, H-32), 0.87 (3H, d, J = 6.8 Hz, H-31). ¹³C NMR (CDCl3, 100 MHz) δ C :170.2 (C-7), 162.3 (C-6), 160.3 (C-4), 139.3 (C-2), 109.0 (C-3), 103.4 (C-5), 1 01.9 (C-1), 98.9 (C-25), 98.8 (C-20), 78.8 (C-17), 76.8 (C-16), 74.6 (C-9), 37.0 ( C-19), 35.6 (C-30), 32.0 (C-26), 31.8 (C-22), 31.5 (C-10), 27.5 (C-21), 27.2 (C-15), 23.5 (C-23), 23.0 (C-27), 21.0 (C-12), 18.9 (C-28), 17.0 (C-32), 16.8 (C-31). Based on the above ¹H NMR and ¹³C NMR spectra, this compound is presumed to be a mixed-origin terpene compound. By comparing with data from the literature (Phytochemistry, 2025, 236, 114493), the compound is identified as talaromyolide C.

[0046] Compound 9 is a yellow powdery solid with the molecular formula C9. 27 H 34 O9. ¹H NMR (CDCl3, 400 MHz) δ H: 6.49 (1H, s, H-5), 6.07 (1H, d, J = 2.0 Hz, H-10), 4.73 (1H, m, H-9), 2.71 (2H, overlap, H-15), 2.46 (1H, m, H-22), 2.12 (3H, s, CH3CO), 2.00 (1H, m, H-27), 1.9 3 (2H, m, H-21, H-26), 1.87 (1H, m, H-27), 1.71 (1H, m, H-30), 1.70 (2H, m, H-21, H-22), 1.73 (1H, m, H-26), 1.46 (3H, d, J= 6.4 Hz, H-12), 1.26 (3H, s, H-23), 1.06 (3H, s, H-28), 0.86 (3H, d, J = 7.2 Hz, H-32), 0.85 (3H, d, J = 7.2 Hz, H-31). ¹³C NMR (CDCl3, 100 MHz) δ C : 170.4 (CH3CO), 169.2 (C-7), 162.2 (C-6), 160.6 (C-4), 136.1 (C-2), 111.0 (C-3), 106 .0 (C-5), 102.0 (C-1), 98.9 (C-20), 98.7 (C-25), 79.2 (C-17), 76.6 (C-16), 75.9 (C-9) 64.3 (C-10), 37.0 (C-19), 35.6 (C-30), 31.9 (C-22), 31.7 (C-26), 27.4 (C-21), 26.8 (C-15), 23.7 (C-23), 22.8 (C-27), 20.7 (CH3CO), 17.0 (C-31), 16.8 (C-32), 16.4 (C-12). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (Tetrahedron, 2020, 76(30): 131349), the compound is identified as talaromyolide E.

[0047] Compound 10 is a white powdery solid with the molecular formula C0. 25 H 32 O7. ¹H NMR (CDCl3, 400 MHz) δ H: 6.36 (1H, s, H-5), 4.68 (1H, m, H-9), 2.89 (1H, dd, J = 16.6, 3.4 Hz, H-10), 2.72 (1H, d, J = 3.2Hz, H-15), 2.61 (1H, dd, J = 16.6, 11.6 Hz, H-10), 2.42 (1H, m, H-27), 2.07 (1H, m, H-22), 2.04 (1H, m, H-26), 1.93 (1H, m, H-21), 1 .77 (1H, m, H-27), 1.76 (1H, m, H-22), 1.75 (1H, m, H-26), 1.74 (1H, m, H-21), 1.54 (3H, d, J = 6.2 Hz, H-12), 1.26 (3H, s, H-23), 1.21 (3H, s, H-30), 1.09 (3H, s, H-31), 1.00 (3H, s, H-28). ¹³C NMR (CDCl3, 100 MHz) δ C :170.3 (C-7), 162.4 (C-6), 159.7 (C-4), 139.5 (C-2), 109.1 (C-3), 103.6 (C-5), 102.2 (C-1), 99.9 (C-25), 79.1 (C-17), 78.6 (C-16), 75.7 (C-20), 74.6 (C-9), 47 .0 (C-29), 40.7 (C-19), 34.1 (C-27), 31.6 (C-10), 30.2 (C-21), 29.7 (C-22), 28. 0 (C-15), 25.6 (C-26), 24.0 (C-23 and C-31), 21.0 (C-12), 19.2 (C-28), 17.9 (C-30). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (Tetrahedron, 2020, 76(30): 131349), the compound is identified as talaromyolide K.

[0048] Compound 11 is a white powdery solid with the molecular formula C11. 27 H 34 O9. ¹H NMR (CDCl3, 400 MHz) δ H: 6.39 (1H, s, H-5), 5.97 (1H, d, J = 2.0 Hz, H-10), 4.67 (1H, m, H-9), 2.78 (1H, d, J = 17.2 Hz, H-15), 2.63 (1H, d, J = 16.8 Hz, H-15), 2.34 (1H, td, J = 13.8, 6.0 Hz, H-22), 2.30 (3H, s, CH3CO), 1.97 (1H, m, H-27), 1.94 (1H, m, H-26), 1.90 (1H, m, H-21), 1.84 (1H, m, H-26), 1.70 (1H, m, H-21), 1.67 (1H, m, H-27), 1.64 (1H, m, H-22), 1.37 (3H, d, J = 6.0 Hz, H-12), 1.29 (3H, s, H-23), 1.16 (3H, s, H-31), 1.12 (3H, s, H-30), 1.00 (3H, s, H-28). ¹³C NMR (CDCl3, 100MHz) δ C : 170.4 (CH3CO), 169.2 (C-7), 162.3 (C-6), 160.1 (C-4), 136.2 (C-2), 111.1 (C-3), 106.1 ( C-5), 102.3 (C-1), 100.0 (C-25), 79.5 (C-17), 78.4 (C-16), 75.9 (C-9), 75.7 (C-20), 64.3 (C-10), 47.1 (C-29), 40.7 (C-19), 34.0 (C-22), 30.9 (C-26), 30.4 (C-21), 29.8 (C-15), 27.7 (C-27), 25.5 (C-30), 24.3 (C-23), 20.7 (CH3CO), 19.3 (C-28), 17.8 (C-31), 16.4 (C-12). Based on the above ¹H NMR and ¹³C NMR spectra, it is inferred that this compound is a mixed-origin terpene compound. By comparing with the data in the literature (JNat Prod, 2024, 87(8): 2034), the compound is identified as talarine H.

[0049] Compound 12 is a yellow oily substance with the molecular formula C12. 26 H 32 O6. ¹H NMR (CDCl3, 400 MHz) δ H: 6.57 (1H, d, J = 10.0 Hz, H-3), 5.81 (1H, d, J = 10.0 Hz, H-2), 5.64 (1H, dd, J = 5.2, 2.0 Hz, H-14), 5.43 (1H, br s, H-23), 4.93 (1H, br s, H-23), 3.75 (3H, s, 26-OCH3), 3.08 (1H, dd, J = 18.8, 2.0 Hz, H-13), 2.56 (1H, dd, J = 13.2, 3.2 Hz, H-13), 2.13 (1H, dd, J = 13.0, 3.2Hz, H-6), 1.84 (1H, t, J = 13.2 Hz, H-6), 1.55 (3H, s, H-24), 1.34 (6H, s, H-17, H-21), 1.30 (3H, s, H-19), 1.25 (3H, s, H-18), 1.14 (3H, s, H-25), 1.03 (1H, dd, J = 13.0, 3.2 Hz, H-5). ¹³C NMR (CDCl3, 100 MHz) δ C :207.6 (C-8), 204.2 (C-1, C-10), 168.6 (C-20), 153.8 (C-3), 145.1 (C-22), 140.4 (C- 15), 126.2 (C-2), 121.0 (C-14), 112.6 (C-23), 80.6 (C-9), 71.7 (C-11), 52.9 (26-OCH 3), 51.2 (C-7), 47.9 (C-16), 45.7 (C-12), 43.3 (C-5), 40.2 (C-6), 39.4 (C-4), 32.1 (C-13), 29.6 (C-17), 28.0 (C-18), 24.3 (C-25), 22.3 (C-24), 18.5 (C-19), 15.2 (C-21). Based on the above ¹H NMR and ¹³C NMR spectra, this compound is presumed to be a mixed-origin terpene. By comparing with data from the literature (Fitoterapia, 2018, 125: 249), this compound is identified as berkeleytrion.

[0050] Compound 13 is a yellow oily substance with the molecular formula C13. 26 H 34 O7. ¹H NMR (DMSO-d6, 400 MHz) δ H: 6.57 (1H, br s, 9-OH), 5.55 (1H, dd, J = 6.2, 2.4 Hz, H-14), 5.24 (2H, br s, H-18, H-23), 4.88 (1H, d, J = 0.8 Hz, H-23), 4.71 (2H, br s, H-18, H-23), 4.59 (1H, d, J = 1.8 Hz, H-18), 3.60 (3H, s, H-26), 2.43 (1H, d, J = 16.2 Hz, H-13), 2.36 (1H, dd, J = 18.0, 6.4 Hz, H-13), 1.84 (1H, dd, J = 12.8, 2.8 Hz, H-6), 1.80 (3H, s, H-17), 1.68 (1H, t, J = 13.6 Hz, H-6), 1.58 (1H, m, H-2), 1.53 (1H, m, H-3), 1.41 (3H, s, H-24), 1.36 (1H, dd, J = 13.6, 5.2 Hz, H-3), 1.21 (3H, s, H-21), 1.19 (3H, s, H-19), 0.90 (1H, dd, J = 13.0, 3.2 Hz, H-5), 0.88 (3H, s, H-25). ¹³C NMR (DMSO-d6, 100 MHz) δ C :209.0 (C-8), 205.2 (C-10), 173.8 (C-1), 169.3 (C-20), 145.6 (C-16), 143.9 (C-22) , 142.5 (C-15), 124.6 (C-14), 114.4 (C-18), 110.8 (C-23), 77.8 (C-9), 71.3 (C-11), 5 2.0 (C-26), 50.5 (C-7), 45.4 (C-12), 41.0 (C-5, C-6), 40.3 (C-4), 33.6 (C-3), 30.9 (C-13), 29.4 (C-2), 26.2 (C-17), 22.3 (C-25), 21.9 (C-24), 16.0 (C-21), 15.9 (C-19). Based on the above ¹H NMR and ¹³C NMR spectra, this compound is presumed to be a mixed-origin terpene. By comparing with data from the literature (Eur J Org Chem, 2018, 1: 48), this compound is identified as berkeleyone C.

[0051] Example 2 Anti-inflammatory activity test

[0052] 1. Initial screening of the activity of ethyl acetate extract: The ethyl acetate extract prepared in Example 1 was tested by the Griess method and found to have an inhibition rate of more than 30% on NO in LPS-induced RAW264.7 cells at a concentration of 500 μg / mL.

[0053] 2. Activity assays of compounds 1-13: The cytotoxicity of all compounds to RAW264.7 cells was first tested using the MTT assay. RAW 264.7 cells were seeded into 96-well plates (1×10⁻⁶ cells / wells). 4 Cells were incubated in 5% CO2 at 37 ℃ for 12 h. Different concentrations of the compound were diluted and applied to the cells. After 24 h, 20 μL (5 mg / mL) MTT solution was added to each well, and after incubation for 4 h, the supernatant was discarded. 150 μL of dimethyl sulfoxide (DMSO) was added to each well for color development. The absorbance (OD) value was measured at 570 nm using a microplate reader. Each experiment was repeated three times. Cell viability (%) = (OD) / (M / mL) 样品 / OD 空白 )×100%. Secondly, the inhibitory effect of the compound on NO induced by LPS in RAW264.7 cells at a non-toxic dose was detected by the Griess method (positive control: dexamethasone). RAW264.7 cells were seeded into 96-well plates (1×10⁻⁶). 5 Cells were cultured in ( / well) for 12 h, after which the culture medium was aspirated, and 100 μL of culture medium (1% FBS) containing different concentrations of drugs was added again. After 1 h, 100 μL of culture medium containing LPS (2 μg / mL) was added for 24 h of treatment. Following the procedure on the Griess kit, 50 μL of supernatant, Griess reagent I, and II were taken and the OD value was measured at 540 nm. The NO content accumulated in the cell supernatant was calculated, and each experiment was repeated three times. The contents of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 10 (IL-10), and interleukin 6 (IL-6) in the cell supernatant were detected by ELISA kit. The results showed that compounds 1-13 exhibited different degrees of inhibitory effects, especially compound 8, which significantly inhibited LPS-induced NO production in RAW264.7 cells at a non-toxic dose (50 μM). Therefore, using the same method as the NO activity assay, cells were treated with the compound and the positive control drug dexamethasone (101.9 μM), the supernatant was collected, and the contents of inflammatory factors (TNF-α, IL-1β, IL-10 and IL-6) were detected according to the instructions on the kit. Each experiment was repeated three times.

[0054] The results showed that compound 8 had a certain inhibitory effect on LPS-induced NO production in RAW264.7 cells. Figure 4The IC50 value was 60.01 µM, which was stronger than the positive control dexamethasone (IC50 value). 50 = 136.84 µM), and showed no cytotoxicity at 70 µM ( Figure 5 Therefore, compound 8 was selected for research on its anti-inflammatory properties.

[0055] The effect of compound 8 on the expression of inflammatory factors TNF-α, IL-1β, IL-10, and IL-6 in LPS-induced RAW264.7 cells was tested using ELISA. The results showed that compared with the control group, the release of TNF-α, IL-1β, IL-10, and IL-6 was significantly increased in the LPS group, while the positive control (dexamethasone, 70 μM) showed a good inhibitory effect on the release of TNF-α, IL-1β, IL-10, and IL-6. When compound 8 was applied to RAW264.7 cells at a concentration of 70 μM, the release of pro-inflammatory factors TNF-α, IL-1β, IL-10, and IL-6 showed a decreasing trend in a dose-dependent manner. Compound 8, at a concentration of 70 µM, significantly inhibited the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-10, and was stronger than the positive control dexamethasone. Figure 6 ).

Claims

1. A marine fungus 23061902, characterized in that... Its strain preservation number is GDMCC No. 66210.

2. An anti-inflammatory compound or a pharmaceutically acceptable salt thereof, characterized in that... The anti-inflammatory compound has the structure shown in compound 1: 。 3. The ethyl acetate extract of marine fungus 23061902 according to claim 1, characterized in that... The preparation method of the ethyl acetate extract includes the following steps: (1) Marine fungus 23061902 was inoculated into rice culture medium and fermented at room temperature for 30 days to obtain fermentation product; (2) The fermentation product obtained in step (1) is soaked in an equal volume of ethyl acetate for extraction 2-4 times, with an interval of 4-6 days between each extraction. The extracts are combined and concentrated under reduced pressure to obtain the ethyl acetate extract.

4. The use of the marine fungus 23061902 of claim 1 in the preparation of any one or more of compounds 1-13 or the ethyl acetate extract of claim 3; the structures of compounds 1-13 are as follows: 。 5. The use of any one or more of the compounds 1-13 of claim 4 or their pharmaceutically acceptable salts, or the ethyl acetate extract of claim 3, in the preparation of an anti-inflammatory drug.

6. An anti-inflammatory drug composition, characterized in that... The pharmaceutical composition uses any one or more of the compounds 1-13 of claim 4 or their pharmaceutically acceptable salts, or the ethyl acetate extract of claim 3 as the active ingredient.

7. The anti-inflammatory pharmaceutical composition according to claim 6, characterized in that... The pharmaceutical composition may also optionally contain pharmaceutically acceptable excipients.

8. The anti-inflammatory pharmaceutical composition according to any one of claims 6-7, characterized in that... The pharmaceutical composition may also optionally contain other anti-inflammatory active ingredients.

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