Chromone compound with anti-inflammatory activity as well as preparation method and application thereof

Abstract

The invention provides a chromone compound with anti-inflammatory activity as well as a preparation method and application thereof. According to the invention, the endophytic fungi Phomopsis asparagi DHS-48 and Pestalotiopsis sp.HHL101 in mangrove forest are subjected to co-culture, and a new chromone compound Phaseolorin J is separated from the co-culture fermentation extract of the endophytic fungi Phomopsis asparagi DHS-48 and Pestalotiopsis sp.HHL101. The structural formula of the chromone compound is shown in the formula (1), the chromone compound is named as Phaseolorin J: # imgabs0 #, it is found through research that Phaseolorin J can play a role in treating RAW 264.7 macrophage inflammatory response stimulated by LPS, and the chromone compound Phaseolorin J can be applied to preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and particularly to a chromone compound having anti-inflammatory activity, a preparation method thereof, and an application thereof. Background Art

[0002] Inflammation is a complex biological response to injury, infection, or tissue damage, involving various immune cells, chemical messengers, and other inflammatory mediators. However, exaggerated inflammatory responses can lead to the development of various chronic diseases, including asthma, cardiovascular diseases, osteoporosis, cancer, obesity, and bronchitis. Under external stimuli, immune cells such as macrophages play a key role in the inflammatory response by releasing inflammatory mediators (such as NO, prostaglandins, iNOS, COX-2, and inflammatory cytokines). The activation of iNOS and COX-2, which affect platelet aggregation, vascular permeability, and thrombus formation, leads to the activation of the NO and PGE2, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways in stimulated cells, regulating the expression of iNOS, COX-2, and the secretion of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. In addition, the activation of the nucleotide-binding oligomerization domain (NOD-)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome multiprotein complex enhances the inflammatory response by regulating the release of pro-inflammatory cytokines; IL-18 and IL-1β in stimulated macrophages.

[0003] LPS is a major component of the outer cell membrane of Gram-negative bacteria and can trigger host inflammatory responses by increasing the production of cytokines, chemokines, and other pro-inflammatory mediators. When macrophages are exposed to LPS during a microbial infection, they initiate the production of inflammatory mediators, subsequently leading to an inflammatory response. Therefore, inhibiting macrophage stimulation is a key therapeutic strategy for treating inflammatory diseases. Summary of the Invention

[0004] In view of this, the present invention provides a chromone compound having anti-inflammatory activity, a preparation method thereof, and an application thereof.

[0005] The inventors isolated a series of cytochalasin and chromone compounds from the endophytic fungus Phomopsis asparagi DHS48 of the mangrove plant Rhizophora apiculata. The present invention co-cultured the mangrove endophytic fungi Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL101, and isolated a new chromone compound, Phaseolorin J, from the co-culture fermentation extract.

[0006] Technical solution of the present invention: A chromone compound, the structural formula of the chromone compound is shown in (1), named Phaseolorin J:

[0007]

[0008] The present invention also provides a method for preparing the chromone compound, comprising the following steps:

[0009] (1) Co-culturing and fermenting Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL101 to obtain a culture fermentation product;

[0010] (2) Extracting the culture fermentation product three times with ethyl acetate, and obtaining a crude extract after the filtrate is evaporated under reduced pressure;

[0011] (3) Mixing the crude extract obtained in step (2) with 85% - 95% v / v methanol-water, adding an equal volume of petroleum ether for extraction three times, removing the petroleum ether layer, and concentrating under reduced pressure to obtain an extract;

[0012] (4) Thoroughly grinding and mixing the extract obtained in step (3) with 100 - 200 mesh normal-phase silica gel powder, using CH2Cl2-MeOH as the eluent, gradient eluting a silica gel column, concentrating under reduced pressure, and after detecting by thin-layer chromatography dot plate, combining similar components to obtain 5 fractions, namely Fr.1 - Fr.5; Fr.5 is subjected to silica gel column and isocratically eluted with CH2Cl2-EtOAc to obtain 6 fractions, namely Fr.5.1 - Fr.5.6;

[0013] (5) Purifying Fr.5.3 using a Sephadex LH-20 column, and then performing reverse-phase HPLC analysis to obtain the target chromone compound.

[0014] Furthermore, in step (1), the culture conditions are as follows: using a petri dish containing potato dextrose agar, the culture temperature is 27 - 29 °C, and the culture time is 4 - 6 days.

[0015] Furthermore, in step (1), the fermentation conditions are as follows: using a rice medium, calculated by weight ratio, the medium formula is: 90 - 110 g of rice, 0.5 - 0.7 g of peptone, 2 - 4 g of crude sea salt, 80 - 120 mL of water; the fermentation temperature is 27 - 29 °C, and the fermentation time is 28 - 32 days.

[0016] Furthermore, in step (3), the material-liquid ratio of the crude extract to methanol-water is 90:0.8 - 1.2 g / L.

[0017] Furthermore, in step (4), the mass ratio of the silica gel powder to the extract is 1.5 - 2:1.

[0018] Further, in step (4), the elution gradient volume ratio of CH2Cl2-MeOH is 30:1, 25:1, 20:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1. The elution volume of each gradient is 4 L, and it is collected every 1 L.

[0019] Further, in step (4), the elution volume ratio of CH2Cl2-EtOAc is 0.9-1.1:0.9-1.1, preferably 1:1.

[0020] Further, in step (5), the purification solvent is prepared by mixing CH2Cl2 and MeOH with a volume ratio of 0.9-1.1:0.9-1.1; the elution solvent for reverse-phase HPLC is prepared by mixing MeOH and H2O with a volume ratio of 38-42:58-62.

[0021] Use of the chromone compound described in the present invention in the preparation of anti-inflammatory drugs.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention co-cultures the mangrove endophytic fungi Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL101, and isolates a new chromone compound Phaseolorin J from the co-culture fermentation extract.

[0024] Moreover, through its pharmacological activity tests, it was shown that Phaseolorin J has a protective effect on the inflammatory response stimulated by lipopolysaccharide (LPS) in RAW 264.7 macrophages. The research results indicate that Phaseolorin J increases cell viability and simultaneously dose-dependently reduces the production of intracellular reactive oxygen species in LPS-stimulated RAW 264.7 macrophages. Phaseolorin J also inhibits NO production. In addition, Phaseolorin J downregulates the expression of IL-1β, IL-6, IL-18, and TNF-α by regulating Nrf2 signaling. The NLRP3 inflammasome protein complex, including NLRP3, ASC, and caspase-1, and the subsequent release of pro-inflammatory cytokines such as IL-1β and IL-18 in LPS-stimulated RAW264.7 macrophages are inhibited by Phaseolorin J in LPS-stimulated RAW 264.7 macrophages. The cytoprotective effect of Phaseolorin J is indicated by the activation of the Nrf2 / HO-1 signal, which is significantly reduced after ML385 inhibits Nrf2 activity. Overall, the research of the present invention found that Phaseolorin J can play a therapeutic role in the inflammatory response of LPS-stimulated RAW 264.7 macrophages, and the chromone compound Phaseolorin J of the present invention can be applied to the preparation of anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 . HPLC chromatograms of the EtOAc extracts from (A) co-culture of DHS-48 and HHL-101 and (B) single culture of DHS-48 and (C) single culture of HHL-101;

[0026] Figure 2 . HPLC chromatogram of Phaseolorin J;

[0027] Figure 3 . 1 1H-NMR of phaseolorin J;

[0028] Figure 4 . 13 13C-NMR of phaseolorin J;

[0029] Figure 5 . DEPT of phaseolorin J;

[0030] Figure 6 . 1 1H- 1 1H-1H COSY of phaseolorin J;

[0031] Figure 7 . HSQC of phaseolorin J(1);

[0032] Figure 8 . HMBC of phaseolorin J(1);

[0033] Figure 9 . NOSEY of phaseolorin J(1);

[0034] Figure 10 . HR-ESI-MS of phaseolorin J(1);

[0035] Figure 11 . Toxic effect of phaseolorin J on Raw264.7 cells;

[0036] Figure 12 . Inhibitory effect of phaseolorin J on NO production in LPS-stimulated Raw264.7 cells;

[0037] Figure 13 . Effect of phaseolorin J on the secretion of inflammatory factor interleukin-1β (IL-1β) in LPS-induced RAW264.7 cells;

[0038] Figure 14 . Effect of phaseolorin J on the secretion of inflammatory factor interleukin-6 (IL-6) in LPS-induced RAW264.7 cells;

[0039] Figure 15 . Effect of phaseolorin J on the secretion of inflammatory factor tumor necrosis factor-α (TNF-α) in LPS-induced RAW264.7 cells;

[0040] Figure 16 . Effect of phaseolorin J on the oxidative stress factor SOD in LPS-induced RAW264.7 cells;

[0041] Figure 17 . Effect of phaseolorin J on the oxidative stress factor MDA in LPS-induced RAW264.7 cells;

[0042] Figure 18 . Flow cytometry diagram of the oxidative stress factor ROS in phaseolorin J-treated LPS-induced RAW264.7 cells;

[0043] Figure 19Flow cytometry data of Phaseolorin J on the oxidative stress factor ROS in LPS-induced RAW264.7 cells;

[0044] Figure 20 Effect of Phaseolorin J pretreatment on the expression of NRF2 in LPS-induced RAW264.7 cells;

[0045] Figure 21 . Inhibition of Phaseolorin J on the expression of IL-18 in LPS-induced RAW264.7 cells;

[0046] Figure 22 . Phaseolorin J inhibits the NLRP3 inflammasome in LPS-induced RAW264.7 cells through NRF2. Detailed implementation mode

[0047] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0048] The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0049] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels unless otherwise specified.

[0050] Example 1 Preparation of chromone compounds

[0051] 1. Fungal source

[0052] The endophytic fungus Phomopsis asparagi DHS-48 was isolated from the fresh root hypocotyls of the Chinese mangrove plant Rhizophora mangle collected from Dongzhai Harbor Mangrove Park, Hainan Island, China using PDA medium. The fungus (strain number: DHS-8) was identified by molecular biology methods (including DNA amplification and ITS region sequencing). The resulting sequence was assigned to GenBank accession number: MT126606. Phomopsis asparagi was deposited in the China Center for Type Culture Collection, deposit number CCTCC NO: M2021318, and the deposit date was March 31, 2021. This fungus has been disclosed in patent CN113603629A.

[0053] The endophytic fungus Pestalotiopsis sp. HHL101 was isolated from fresh branches of the Chinese mangrove plant Rhizophora stylosa collected from Dongzhai Harbor Mangrove Park, Hainan Island, China, using PDA medium. The fungus (strain number HHL-101) was identified by molecular biology methods (including DNA amplification and ITS region sequencing). The resulting sequence was deposited in GenBank under accession number: EF451799.

[0054] 2. Isolation method

[0055] (1) First, a single colony of Phomopsis asparagi DHS-48 or Pestalotiopsis sp. HHL101 was inoculated into a Petri dish containing potato dextrose agar (PDA) and cultured at 28 °C for 5 days. Subsequently, a 1 cm² single colony of DHS-48 and HHL-101 was inoculated into a flask (160 × 1 L), with 100 g of rice, 0.6 g of peptone, 3 g of crude sea salt, and 100 mL of water added to each flask. The mixture was sterilized at 121 °C for 30 min and fermented at 28 °C for 30 days to obtain a culture fermentation product.

[0056] (2) The culture fermentation products in 160 flasks were extracted three times with 400 mL of EtOAc (ethyl acetate). After the filtrate was evaporated under reduced pressure, 90 g of crude extract was obtained.

[0057] (3) To remove fatty acid impurities from the crude extract, the crude extract was mixed with 1 L of 90% v / v methanol-water, and extracted three times with an equal volume of petroleum ether. The petroleum ether layer was removed, and the extract was concentrated under reduced pressure to obtain 70 g of extract.

[0058] (4) The extract was thoroughly ground and mixed with 100-200 mesh normal-phase silica gel powder (silica gel powder mass: extract mass = 1.5:1), and CH2Cl2-MeOH (dichloromethane and methanol) was used as the eluent to elute the silica gel column with a gradient elution. The elution gradient was 30:1, 25:1, 20:1, 15:1, 10:1, 8:1, 6:1, 3:1, 1:1, v / v) (the elution volume for each gradient was 4 L, collected every 1 L, and a total of 36 sub-fractions were obtained after concentration under reduced pressure). After thin-layer chromatography (TLC) plate detection, similar components were combined to obtain 5 fractions (Fr.1-Fr.5). Fr.5 was eluted isocratically with CH2Cl2-EtOAc (1:1, v / v) using a silica gel column. After thin-layer chromatography (TLC) plate detection, similar components were combined to obtain 6 fractions (Fr.5.1-Fr.5.6).

[0059] (5) Purify Fr.5.3 using a Sephadex LH-20 column (CH2Cl2 / MeOH, 1:1, v / v), and then perform reverse-phase HPLC (MeOH-H2O 40:60, v / v) analysis to obtain compound Phaseolorin J (20 mg).

[0060] 3. Analysis of secondary metabolites from co-culture large-scale fermentation

[0061] Perform HPLC chromatographic analysis (HPLC-UV) on the ethyl acetate crude extract of the rice medium co-cultured with DHS-48 and HHL-101 for 30 days, and compound Phaseolorin J (20 mg) was found ( Figure 1 A), and this compound was not detected in the single medium ( Figure 1 B, C). In previous laboratory work, the target strain DHS-48 was regulated by the epigenetic inhibitor sodium butyrate, and a new compound Phaseolorin J (15 mg) was found, and Phaseolorin J can moderately inhibit the proliferation of ConA-induced T lymphocytes and LPS-induced B lymphocytes. The yield of Phaseolorin J under co-culture conditions of DHS-48 is 1.3 times that under the regulation of the epigenetic inhibitor sodium butyrate.

[0062] As Figure 1 shown, HPLC chromatograms of the EtOAc extracts from (A) co-culture of DHS-48 and HHL-101, (B) single culture of DHS-48, and (C) single culture of HHL-101. C18 chromatographic column (Agilent Technologies, 10 mm × 250 mm). Solvents: A, H2O; B, MeOH. Linear gradient: 0 min, 10% B; 70 min, 80% B. Flow rate 2 mL / min. UV detection, wavelength λ = 210 nm.

[0063] 4. Compound purity analysis

[0064] HPLC chromatograms used to detect the purity of the compound. HPLC chromatogram: C18 chromatographic column (Agilent Technologies, 10 mm × 250 mm). Solvents: A, H2O; B, MeOH. Linear gradient: 0 min, 10% B; 70 min, 80% B. Flow rate 2 mL / min. UV detection, wavelength λ = 210 nm.

[0065] The HPLC chromatogram of the compound is as Figure 2 shown.

[0066] 5. Compound information

[0067] Phaseolorin J: Pale yellow amorphous powder; [α] 20D + 160(c 0.0001, MeOH); UV (MeOH) λmax 214 nm. 1 1H NMR (400 MHz, CD3OD) δ 1.86 (s, CH3-11), 4.03 - 4.12 (H a 4.12, d, 13.2; H b 4.03, d, 13.2, 2H), 4.55 (d, 4.8, 1H), 4.69 (s, 1H), 5.61 (q, 1.7, 1H), 6.43 (d, 8.2, 1H), 6.52 (d, 8.2, 1H), 7.38 (t, 8.2, 1H). 13 13C NMR (101 MHz, CD3OD) δ 19.2 (CH3-11), 64.1 (CH2-12), 67.9 (CH-8), 74.3 (CH-5), 74.5 (C-10a), 86.5 (C-8a), 108.4 (C-9a), 108.7 (CH-4), 109.3 (CH-2), 122.3 (CH-7), 138.9 (CH-3), 140.5 (C-6), 160.8 (C-4a), 163.2 (C-1), 197.2 (C-9), HRESIMS m / z 331.0780 [M+Na] + (calcd for C 15 H 16 O7Na 331.0788).

[0068] As Figure 3 shown, 1 1H-NMR of phaseolorin J;

[0069] As Figure 4 shown, 13 13C-NMR of phaseolorin J;

[0070] As Figure 5 shown, DEPT of phaseolorin J;

[0071] As Figure 6 shown, 1 1H- 1 1H COSY of phaseolorin J;

[0072] As Figure 7 shown, HSQC of phaseolorin J;

[0073] As Figure 8 shown, HMBC of phaseolorin J;

[0074] As Figure 9 shown, NOSEY of phaseolorin J;

[0075] As Figure 10 shown, HR-ESI-MS of phaseolorin J.

[0076] In summary, the structural formula of Phaseolorin J is

[0077]

[0078] Experimental Example

[0079] This study was designed by hypothesizing that Phaseolorin J inhibits the inflammatory response by regulating the expression of inflammatory mediators in LPS-stimulated RAW264.7 macrophages.

[0080] 1. Materials and Methods

[0081] 1.1 Experimental Materials

[0082] The mouse monocyte macrophage cell line RAW264.7 cells were purchased from Wuhan Punosai Life Science Co., Ltd.

[0083] 1.2 Instruments and Reagents

[0084] Main experimental instruments: laminar flow hood (AIRTECH); carbon dioxide cell incubator (Galaxy R); inverted microscope (OLYMPUS); microplate reader (KHB-ST-360); ultrapure water instrument (Biotech); autoclave (HVE-50); ultra-low temperature freezer (MDF-593(N)); laser confocal microscope (TCS-SP5) -20°C refrigerator (Haier); low-speed centrifuge (ThermoFisher); cryogenic high-speed centrifuge (Thermo Fisher); shaker; metal bath heater; incubator; water bath.

[0085] Main experimental reagents: Fetal bovine serum (FBS), penicillin / streptomycin, and DMEM medium were all purchased from Wuhan Punosai Life Science Co., Ltd. Lipopolysaccharide (Sigma); dimethyl sulfoxide (Amresco); Cell Counting Kit-8 (CCK-8) detection kit, propidium iodide (PI); IL-18 ELISA kit; SOD kit, MDA kit, ROS kit (Beyotime) were purchased from Beyotime Biotechnology Co., Ltd.; enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were purchased from Shanghai Fusheng Industrial Co., Ltd.; polyclonal antibodies against NLRP3 and NRF2 and HRP-labeled immunoglobulin G (IgG) were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0086] 1.2 RAW264.7 cell culture and treatment

[0087] (1) Cell resuscitation

[0088] 1) Take out the cryopreservation tube from liquid nitrogen, quickly place it in a 37°C water bath, and take it out after the ice has completely melted.

[0089] 2) Quickly transfer the melted cell cryopreservation tube to the laminar flow hood, transfer the cells to a 15 mL centrifuge tube, and add 5 mL of complete medium and mix well.

[0090] 3) Centrifuge at 300 g or 1200 rpm for 3 min, and discard the supernatant.

[0091] 4) Resuspend the cell pellet with 5 ml of complete medium, then inoculate it into a T25 culture flask with a breathable cap, and place it in a 37°C CO2 incubator for culture.

[0092] (2) Cell culture

[0093] RAW264.7 cells were cultured in high-glucose DMEM medium containing 1% double antibody and 10% fetal bovine serum, and placed in a 37°C, 95% O2 and 5% CO2 incubator for culture. In the subsequent experimental process, the cells were cultured for 24 h before drug treatment and then the experiment was carried out.

[0094] RAW 264.7 macrophages were cultured in a humidified incubator containing 5% CO at 37°C, and DMEM supplemented with 10% FBS and 1% penicillin / streptomycin mixture was used to maintain RAW 264.7 macrophages. Subculture was performed every two days until they reached exponential growth suitable for in vitro experiments.

[0095] (3) Drug treatment

[0096] Before the experiment, Phaseolorin J was diluted with PBS to configure final treatment concentrations of 20, 40, and 60 μM. Throughout the study, cells were stimulated with LPS at a concentration of 0.1 μg / mL.

[0097] 1.3 CCK-8 assay for cell viability

[0098] According to the method of previous studies, the effect of Phaseolorin J on cell viability was evaluated using cck-8 assay. 10 4 Cells / well were seeded in 96-well plates and incubated for 24 hours. Before stimulation with LPS, cells were pretreated with various concentrations of Phaseolorin J and then incubated at 37°. CCK-8 reagent was added respectively, and the absorbance (A value) at 450 nm of each well was detected using a microplate reader after 2 h.

[0099] 1.4 Determination of oxidation and antioxidant-related indices

[0100] To explore the changes in intracellular oxidation and antioxidant indices in LPS-stimulated RAW 264.7 macrophages by Phaseolorin J. After incubation for 24 hours, cells were treated with Phaseolorin J sample doses and incubated for 1 hour. Then cells were stimulated with LPS. After 6 hours of cell stimulation, DCF-DA solution was added to the cells, and ROS was measured using a flow cytometer. The assay kits for SOD (hydroxylamine method) and MDA (colorimetric method) in cells were purchased from Beyotime Biotechnology Company, and the specific operation procedures were carried out according to the kit instructions.

[0101] 1.5 Measurement of NO production

[0102] NO was produced in LPS-stimulated RAW 264.7 macrophages using the Griess assay. Pretreatment was carried out with Phaseolorin J sample concentrations, and then cells were stimulated with LPS for 6 hours. Subsequently, Griess reagent was mixed with the cell supernatant in each well at room temperature for 10 minutes. The absorbance at 540 nm was measured using a microplate reader.

[0103] 1.6 ELISA analysis

[0104] The cell culture media in the multi-well plates of all RAW 264.7 macrophage groups were collected. The production of inflammatory cytokines in the collected cell culture media was analyzed using relevant ELISA detection kits. The enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were purchased from Shanghai Fusen Industrial Co., Ltd. The absorbance at 450 nm was measured using a microplate reader. The samples were normalized using a standard curve. The specific operation procedures were carried out according to the kit instructions.

[0105] 1.7 Immunofluorescence assay

[0106] RAW 264.7 macrophages were cultured in a confocal dish and incubated for 24 h before sample treatment. After 30 minutes of LPS stimulation, the wells were washed with PBS and fixed with 4% paraformaldehyde. Before incubating overnight with the primary antibody, the cells were blocked with blocking buffer for 1 hour. Then the cells were treated with the corresponding secondary antibody. After washing with PBS, the cells were treated with an anti-quenching reagent containing DAPI for 5 minutes, and the protein expression in the cells was observed using a laser confocal microscope.

[0107] 1.8 Statistical analysis

[0108] One-way ANOVA was performed using IBM SPSS Statistics 26 for data comparison among multiple groups. P < 0.05 and P < 0.001 were considered statistically significant differences. GraphPad Prism 9.5.0 software was used to plot the data information.

[0109] 2 Results and discussion

[0110] 2.1 Effect of Phaseolorin J on the viability of RAW264.7 cells

[0111] Here, we used the CCK-8 method to determine the safe drug concentration range of Phaseolorin J on RAW264.7 cells. The results showed that compared with the control group, Phaseolorin J had no significant effect on cell viability when the concentration was within 50 μmol / L, and the cell survival rate was about 60% when the concentration was 150 μmol / L. As Figure 11 shown, the cytotoxicity IC 50 of the compound Phaseolorin J on RAW264.7 cells was 194.7 μM.

[0112] 2.2 Effect of Phaseolorin J on the NO expression of RAW264.7 cells

[0113] As a major component of the bacterial cell wall, LPS stimulates cells to produce inflammation, thereby generating a large amount of nitric oxide. As a typical inflammatory effector, NO causes immune damage to cells and exacerbates cell inflammation. The release amount of NO was detected. As Figure 12 the results showed, compared with the LPS group, the release amount of NO decreased significantly with the increase in the concentration of Phaseolorin J, showing a dose-dependent manner. There was a downward trend when the concentration was 30 μmol / L, and when the concentration was 60 μmol / L, the release amount of NO was only about 50%. The IC 50 was 63.15 μΜ.

[0114] 2.3 Effects of Phaseolorin J on the secretion of inflammatory factors in LPS-induced RAW264.7 cells

[0115] The effects of Phaseolorin J on the secretion of inflammatory factors in LPS-induced RAW264.7 cells are shown in Figure 13 、 14 、and Figure 15.

[0116] LPS stimulation causes a typical NF-κB signaling pathway, triggering the expression of downstream NLRP3 inflammasome-related proteins and the release of TNF-α. NLRP3 inflammasome assembly and activation are induced by ATP, and through the NLRP3 / Caspase-1 / IL-1β signaling pathway, it ultimately leads to macrophage inflammation. In this article, to further study how Phaseolorin J affects the NLRP3 inflammasome, the expression levels of inflammatory factors IL-1β, IL-6, and TNF-α in the cell supernatant were detected by ELISA. The results showed that compared with the LPS group, Phaseolorin J significantly inhibited the release of several inflammatory factors such as IL-1β, and the differences among groups were statistically significant (P<0.05).

[0117] 2.3 Phaseolorin J pretreatment alleviates LPS-induced oxidative stress in RAW264.7 cells

[0118] The effects of Phaseolorin J on oxidative stress factors in LPS-induced RAW264.7 cells are shown in Figure 16 、 17 、Figure 18 and Figure 19.

[0119] Compared with the control group, after LPS treatment, the ROS level and MDA activity increased significantly, while the SOD activity decreased significantly (all P<0.05). Compared with the model group, Phaseolorin J treatment led to a significant decrease in the ROS level and MDA activity, and a significant increase in SOD activity (P<0.05). The positive group showed similar effects on the ROS level and the activities of MDA and SOD. Flow cytometry results also showed that Phaseolorin J significantly reduced the ROS level in RAW264.7 cells. Therefore, these results indicate that Phaseolorin J can significantly regulate LPS-induced oxidative stress in RAW264.7 cells.

[0120] Oxidative stress can enhance the expression of pro-inflammatory genes, while inflammatory cytokines can induce the production of ROS. They cross each other and form a vicious cycle. Oxidative stress is caused by the imbalance between excessive ROS and defects in the antioxidant defense system. Our results show that Phaseolorin J inhibits the activation of oxidative stress factors in LPS-induced RAW264.7 cells and reduces ROS production. The above results indicate that Phaseolorin J has significant antioxidant properties.

[0121] 2.4 Effect of Phaseolorin J pretreatment on NRF2 expression in LPS-induced RAW264.7 cells

[0122] One of the characteristics of LPS-induced RAW264.7 inflammation is oxidative stress. Nrf2 signaling is an important pathway to protect cells from inflammation. The effect of Phaseolorin J on Nrf2 expression was analyzed using nuclear translocation. LPS stimulation decreased Nrf2 expression. However, treatment with Phaseolorin J increased the Nrf2 protein level in RAW264.7 cells. Phaseolorin J treatment reduced the total ROS in RAW264.7 cells ( Figure 20 ). Therefore, we speculate that Phaseolorin J may inhibit oxidative stress and thus inflammation by activating the Nrf2 pathway.

[0123] 2.5 Phaseolorin J inhibits the NLRP3 inflammasome in LPS-induced RAW264.7 cells through NRF2

[0124] The protein composition of the NLRP3 inflammasome, including NLPR3, caspase-1, and apoptosis-associated speck-like protein containing a CARD domain (ASC), plays a crucial role in the immune response by regulating the release of pro-inflammatory cytokines IL-1β and IL-18. In this study, the effect of Phaseolorin J on the secretion of IL-18 in LPS-stimulated RAW 264.7 macrophages was measured by ELISA. As Figure 21 shown, compared with the control group, the expression of IL-18 increased after LPS stimulation. However, Phaseolorin J significantly downregulated the expression of IL-18 in a dose-dependent manner. These results indicate that Phaseolorin J has the activity to inhibit the NLRP3 inflammasome.

[0125] As Figure 22As shown, Phaseolorin J pretreatment can partially inhibit the activation of the NLRP3 inflammasome induced by LPS. In RAW264.7 cells pretreated with the Nrf2-specific inhibitor ML385, the NLRP3 inflammasome increased, indicating that a decrease in Nrf2 expression can promote the activation of the NLRP3 inflammasome. In summary, Phaseolorin J significantly downregulates the expression of inflammatory mediators such as NO, and pro-inflammatory cytokines such as IL-18, IL-6, and IL-1β by regulating the NRF2 signaling pathway in LPS-stimulated RAW 264.7 macrophages. Phaseolorin J inhibits the expression of NLRP3 inflammasome molecules in LPS-stimulated RAW 264.7 macrophages. The research results reveal that Phaseolorin J is a potential NLRP3 inflammasome inhibitor by inhibiting the NRF2 pathway.

[0126] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. A chromone compound, characterized in that: The structural formula of the chromone compound is shown in (1):

2. The method for preparing chromone compounds according to claim 1, characterized in that: The following steps are involved: (1) co-culturing and fermenting Phomopsis asparagi DHS-48 and Pestalotiopsis sp. HHL101 to obtain a culture fermentation product; (2) extracting the fermented product from step (1) with ethyl acetate, and evaporating the filtrate under reduced pressure to obtain a crude extract; (3) mixing the crude extract of step (2) with 85% to 95% v / v methanol-water, adding an equal volume of petroleum ether for extraction, removing the petroleum ether layer, and concentrating under reduced pressure to obtain an extract; (4) The extract obtained in step (3) was fully ground and mixed with 100-200 mesh normal silica gel powder, and gradient eluted on a silica gel column using CH2Cl2-MeOH as the eluent. After reduced pressure concentration, 36 small fractions were obtained in total. After thin layer chromatography spot plate detection, similar components were combined to obtain 5 fractions, namely Fr.1-Fr.5; Fr.5 was subjected to a silica gel column and isocratically eluted with CH2Cl2-EtOAc to obtain 6 fractions, namely Fr.5.1-Fr.5.6; (5) Fr.5.3 was purified using a Sephadex LH-20 column and then analyzed by reverse phase HPLC to obtain the target chromone compound.

3. The method for preparing chromone compounds according to claim 2, characterized in that: Step (1), culture conditions: using a culture dish containing potato dextrose agar, the culture temperature is 27-29° C., and the culture time is 4-6 days.

4. The method for preparing chromone compounds according to claim 2, characterized in that: Step (1), fermentation conditions: using rice culture medium, according to the weight ratio, the culture medium formula: rice 90-110g, peptone 0.5-0.7g, coarse sea salt 2-4g, water 80-120mL; fermentation temperature is 27-29°C, and fermentation time is 28-32 days.

5. The method for preparing chromone compounds according to claim 2, characterized in that: In step (3), the liquid-to-crude extract ratio (g / L) to methanol-water is 90:0.8-1.

2.

6. The method for preparing chromone compounds according to claim 2, characterized in that: In step (4), the mass ratio of the silica gel powder to the extract is 1.5 to 2:

1.

7. The method for preparing chromone compounds according to claim 2, characterized in that: In step (4), the gradient volume ratio of CH2Cl2-MeOH for elution was 30:1, 25:1, 20:1, 15:1, 10:1, 8:1, 6:1, 3:1, and 1:1, and the volume of each gradient elution was 4 L, with one step of 1 L.

8. The method for preparing chromone compounds according to claim 2, characterized in that: In step (4), the CH2Cl2-EtOAc elution volume ratio is 0.9-1.1:0.9-1.

1.

9. The method for preparing chromone compounds according to claim 2 or 8, characterized in that: In step (5), the purification solvent is prepared by mixing CH2Cl2 and MeOH in a volume ratio of 0.9-1.1:0.9-1.1; the elution solvent of the reverse phase HPLC is prepared by mixing MeOH and H2O in a volume ratio of 38-42:58-62.

10. Use of the chromone compound according to claim 1 in the preparation of anti-inflammatory drugs.

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