Novel skeleton type cytochalasin compound and application of novel skeleton type cytochalasin compound in preparation of medicine with anti-inflammatory activity

Abstract

The invention relates to the technical field of medical chemistry, and particularly discloses a cytochalasin compound boerelasin L with a novel skeleton type, which is separated from Boeremia exigua of Fritillaria hupehensis. Specifically, the invention relates to an application of the compound in preparation of drugs with significant inhibitory activity on NO inflammatory effect, by establishing an LPS-induced RAW264.7 cell inflammation model, the result shows that the compound has significant anti-inflammatory activity (IC50 = 8.56 [mu] M) and has no cytotoxicity, and by means of ELISA and Western Blot methods, it is found that the compound can down-regulate expression of iNOS and COX-2 and inhibit secretion of inflammatory factors IL-6 and TNF-alpha, so that the compound can be applied to preparation of drugs with significant inhibitory activity on NO inflammatory effect. The cytochalasin compound provided by the invention realizes dose-dependent inhibition of LPS-induced NO generation, and has excellent safety and patent medicine potential at the same time.

Description

Technical Field

[0001] This invention relates to the fields of medicine and chemical technology, and in particular to a cytochalasin compound derived from the endophytic fungus *Boeremia exigua* from *Fritillaria cirrhosa* in Hubei Province and its application in the preparation of drugs with anti-inflammatory activity. Background Technology

[0002] Inflammation is a defensive response to stimuli from both inside and outside the body, and it is a complex physiological and pathological process. Excessive inflammatory responses can lead to tissue damage and disease. Studies have shown that during the inflammatory process, lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, can activate immune cells such as macrophages, promoting the excessive expression of nitric oxide synthase (iNOS) and catalyzing the production of excessive nitric oxide (NO). Excessive NO not only triggers oxidative stress, damaging intracellular proteins, nucleic acids, and other biomolecules, but it can also induce the release of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), forming an inflammatory cascade amplification effect, which is closely related to the pathological processes of various diseases such as diabetes, rheumatoid arthritis, and atherosclerosis. Therefore, the development of compounds that effectively inhibit LPS-induced NO production is of great significance for the treatment of inflammatory diseases.

[0003] Cytochalasin is a class of fungal secondary metabolites with unique tricyclic or polycyclic structures. Due to the diverse functional groups in their structure, including isopentenyl side chains, ketone groups, and hydroxyl groups, they exhibit rich biological activities in antitumor, antibacterial, anti-inflammatory, and immunomodulatory fields, making them a hot topic in natural product chemistry research. In recent years, researchers have continuously isolated novel cytochalasin compounds from fungi of different sources, hoping to explore their potential medicinal value. However, reported cytochalasin compounds suffer from unsatisfactory activity, a single mechanism of action, and high cytotoxicity, limiting their application in drug development. Therefore, there is an urgent need to discover cytochalasin compounds with novel structures and superior activities.

[0004] Fritillaria hupehensis Hsiao et KCHsia, belonging to the genus Fritillaria in the family Liliaceae, has its bulb as the main medicinal part, possessing effects such as clearing heat and resolving phlegm, relieving cough and dissipating nodules. Endophytic fungi, in the process of co-evolution with host plants, can produce unique metabolites to adapt to special ecological environments, providing novel and bioactive compounds that are difficult to obtain through artificial biosynthesis or chemical synthesis. They are an important source of novel structurally active natural products and clinical drugs. To investigate the novel bioactivity of cytochalasin-like compounds in the endophytic fungi of Fritillaria hupehensis, the applicant investigated the anti-inflammatory activity of a novel cytochalasin skeleton isolated from the endophytic fungi of Fritillaria hupehensis. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a novel cytochalasin skeleton type and its application in the preparation of drugs with anti-inflammatory activity.

[0006] A cytochalasin with anti-inflammatory activity has the following structural formula:

[0007]

[0008] Molecular formula: C 30 H 39 NO4

[0009] The name is: boerelasin L

[0010] The method for preparing the cytochalasin includes the following steps: fermentation product of the endophytic fungus *Boeremiaexigua* from *Fritillaria hupehensis* on oat solid medium is extracted by soaking in a chloroform-methanol mixture (preferably chloroform:methanol = 1:1, v / v). The extract is concentrated to remove the solvent, then dissolved in water, and extracted with ethyl acetate. The ethyl acetate portion is concentrated to obtain an extract. The extract is then purified sequentially by normal-phase silica gel column chromatography, medium-pressure preparative liquid chromatography, Sephadex LH-20 gel column chromatography, normal-phase silica gel column chromatography, and high-performance liquid chromatography to obtain the compound.

[0011] Furthermore, the elution conditions for the normal phase silica gel column chromatography are as follows: the eluent is dichloromethane-methanol (1:0, 20:1, 10:1, 5:1, 0:1, v / v).

[0012] Furthermore, the elution conditions for the medium-pressure liquid preparative chromatography are as follows: the packing material is RP-18 (20-45 μm), and the eluent is methanol-water (50:50, 60:40, 70:30, 80:20, 90:10, v / v).

[0013] Furthermore, the elution conditions for the Sephadex LH-20 gel column chromatography are as follows: the mobile phase is methanol, and the amount of methanol used is 10 column volumes.

[0014] Furthermore, the elution conditions for the normal phase silica gel column chromatography are as follows: the eluent is dichloromethane-methanol (40:1, 20:1, 10:1, 5:1, 0:1, v / v).

[0015] Furthermore, the elution conditions for the high performance liquid chromatography are as follows: C18 column (9.4 mm × 150 mm, 5 μm), mobile phase is acetonitrile-water (65:35-75:25, v / v), elution time is 30 min, flow rate is 4 mL / min, and detection wavelength is 205 nm.

[0016] Furthermore, the oat solid culture medium ferment of Boeremia exigua was prepared by the following method: Boeremia exigua was inoculated on PDA medium and grown for 8 days under ambient temperature and light conditions. Then, bacterial blocks were picked from the PDA medium and inoculated onto oat solid culture medium (oats and distilled water were mixed at a ratio of 1g:1mL and placed in a culture bottle, and sterilized at 120℃ for 20min) for fermentation culture. After culturing at 25℃ in the dark for 30 days, the oat solid culture medium ferment of Boeremia exigua was obtained.

[0017] The application of cytochalasin in the preparation of drugs that can inhibit NO production is also within the scope of protection of this invention.

[0018] The use of the aforementioned cytochalasin derivatives in the preparation of drugs that downregulate iNOS and / or COX-2 expression levels also falls within the scope of protection of this invention.

[0019] The use of the cytochalasin in the preparation of drugs that inhibit the secretion of inflammatory factors IL-6 and / or TNF-α is also within the scope of protection of this invention.

[0020] Another object of the present invention is to provide a medicament for the application of the cytochalasin in the preparation of a drug for diseases related to metabolism, immune regulation and / or signal transduction caused by excessive levels of NO at physiological levels.

[0021] Another object of the present invention is to provide an anti-inflammatory drug, wherein the anti-inflammatory drug has cytochalasin as the main active ingredient.

[0022] The drugs described above include various acceptable dosage forms, preferably tablets, powders, capsules, oral liquids, emulsions, ointments, creams, injections, suspensions, tinctures, granules, or aerosols. All of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0023] In this invention, the anti-inflammatory activity is specifically manifested in its ability to significantly reduce the NO inflammatory effect induced by LPS in mouse mononuclear macrophage leukemia cells (RAW264.7). Experiments of this invention demonstrate that the cytochalasin at a concentration of 40 μM inhibits NO production by 91.86%, and its IC50 value is [missing information]. 50 =8.56 μM, and non-cytotoxic. Studies using ELISA (enzyme-linked immunosorbent assay) and Western blotting revealed that the cytochalasin inhibited the expression of inflammatory factors COX-2, iNOS, IL-6, and TNF-α in a concentration-dependent manner, demonstrating its potential use in the preparation of novel anti-inflammatory drugs.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] 1. This invention discloses a novel cytochalasin with a skeletal type derived from the endophytic fungus Boeremia exigua from Fritillaria thunbergii in Hubei Province. Unlike the known compound cytochalasin T, its C-5 methyl group is replaced by an ethyl group.

[0026] 2. This invention provides a cytochalasin with anti-inflammatory activity that can significantly reduce the NO inflammatory effect induced by LPS in RAW264.7 cells, and has the potential use in the preparation of novel anti-inflammatory drugs.

[0027] 3. The cell relaxin for which this invention is intended to be protected can be obtained by extraction and purification from plant endophytic fungi, and has advantages such as short culture cycle, high operational feasibility, no chemical pollution, and green environmental protection. Attached Figure Description

[0028] Figure 1 The hydrogen spectrum (600 MHz, CDCl3) of the compound prepared in Example 1;

[0029] Figure 2 The carbon spectrum (150MHz, CDCl3) and DEPT spectrum of the compound prepared in Example 1 are shown.

[0030] Figure 3 The HMQC diagram of the compound prepared in Example 1 is shown below.

[0031] Figure 4 The HMBC diagram of the compound prepared in Example 1;

[0032] Figure 5 The compound prepared in Example 1 1 H- 1 H COSY diagram;

[0033] Figure 6 The ROESY graph of the compound prepared in Example 1 is shown below.

[0034] Figure 7 The NO inhibition diagram is shown for the compound prepared in Example 1 of Example 3;

[0035] Figure 8 The effect of the compound prepared in Example 1 of Example 3 on the inflammatory factors IL-6 and TNF-α (n=3, i.e., 3 ELISA tests); Note: Compared with the blank group, ### P<0.001; Compared with the LPS group, **P<0.01,***P<0.001;

[0036] Figure 9 The effect of the compounds prepared in Example 1 of Example 3 on the expression of COX-2 and iNOS proteins (n=3, i.e., 3 protein electrophoresis experiments); Note: Compared with the blank group, ### P<0.001; compared with the LPS group, **P<0.01,***P<0.001. Detailed Implementation

[0037] To make the purpose and content of this invention clearer, the applicant will now provide a clear and complete description of the technical solution of this invention in conjunction with specific embodiments.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1:

[0041] The endophytic fungus *Boeremia exigua* from *Fritillaria hupehensis* was isolated from the roots of fresh *Fritillaria hupehensis* plants. The *Fritillaria hupehensis* was collected from Shuanghe Community, Xintang Township, Enshi City, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province. The strain was self-numbered as Bei 21. The ITS sequence determination results showed that the maximum similarity between this sequence and *Boeremia exigua* was as high as 100%. Gene bank accession number: MT154621.1. Therefore, it was identified as *Boeremia exigua*. This strain is deposited in the Microbial Culture Bank of the School of Pharmacy, South-Central University for Nationalities (published, see: Lü Xiao, Ye Ke, Ma Xujun, et al. Study on chemical composition and anti-inflammatory activity of endophytic fungus *Boeremia exigua* from *Fritillaria hupehensis* [J]. Journal of South-Central University for Nationalities (Natural Science Edition), 2022, 41(2): 174-179).

[0042] The endophytic fungus *Boeremia exigua* from *Fritillaria hupehensis* was cultured using oat-based solid-state fermentation for large-scale propagation. The test tubes containing the preserved strain were removed from the refrigerator and placed in a sterile environment at room temperature for 1 hour. Then, 5mm diameter mycelial blocks were inoculated onto PDA agar plates under sterile conditions. After 8 days of growth at room temperature in the dark, 5mm diameter mycelial blocks were picked from the PDA plates and inoculated onto oat-based agar plates. A total of 255 bottles were cultured under the following conditions: 30 days in the dark at 25℃. The oat-based culture medium consisted of 50g oats / bottle and 50mL distilled water / bottle, placed in a 500mL culture flask and sterilized at 120℃ for 20 minutes.

[0043] The isolation and purification process of cytochalasin, the compound described in the claims and specification, is as follows: Oat solid culture medium fermented for 30 days was collected, crushed, and soaked five times in a mixed reagent (chloroform:methanol = 1:1, v / v), each soaking time being 24 hours. After each soaking, the extracts were centrifuged, and the extracts from the five centrifugations were combined. The solvent in the extract was removed by vacuum concentration, and water was added to dissolve the extract to 7 L. The extract was then extracted five times with ethyl acetate, and the ethyl acetate fractions were combined. The crude extract was concentrated under reduced pressure to obtain a total of 315.33 g. Gradient elution was performed using 80-100 mesh normal-phase silica gel column chromatography (CH2Cl2-MeOH = 1:0, 20:1, 10:1, 5:1, 0:1, v / v). The fractions were developed and segmented by TLC (developing solvent: chloroform:methanol = 20:1, v / v; thin-layer chromatography silica gel plates, Qingdao Ocean Chemical Plant), yielding a total of eight (AH) fractions.

[0044] Fragment D (eluted from a dichloromethane / methanol volume ratio of 20:1, with eluent used for 5 column volumes, 32.5 g) was subjected to high-performance medium-pressure preparative liquid chromatography (Biotage SP1, reversed-phase packing material: RP-18, 20-45 μm, Fuji Silysia Chemical Ltd., Japan) with a gradient elution of methanol-water systems (methanol / water = 50:50, 60:40, 70:30, 80:20, 90:10, v / v) at a flow rate of 20 mL / min. The elution time for each of the five ratios was 60 min. The fractions were then detected and visualized by thin-layer chromatography (developing solvent: petroleum ether: acetone = 4:1, v / v). Similar or identical fractions were combined to obtain 10 subfractions, which were labeled D1-D10 in ascending order of polarity. Fragment D7 (23.5 g, eluted with methanol / water at a volume ratio of 70:30) was eluted with methanol (10 column volumes) using a Sephadex LH-20 gel column chromatography (Pharmacia Fine Chemical Co., Ltd., Sweden). Thin-layer chromatography (TLC) was then performed, and similar or identical fragments were combined to obtain six subfractions, labeled D7-1 to D7-6 in ascending order of polarity. Fragment D7-3 (6.8 g), eluted with the third column volume, was subjected to gradient elution with a 200-300 mesh normal-phase silica gel column in a dichloromethane-methanol system (CH2Cl2-MeOH = 40:1, 20:1, 10:1, 5:1, 0:1, v / v) to obtain seven subfractions, D7-3-1 to D7-3-7. Component D7-3-4 (31.5 mg, eluted with dichloromethane / methanol at a volume ratio of 10:1, eluent volume of 3 column volumes) was then subjected to high performance liquid preparative chromatography (Agilent 1260; column: Agilent Zorbax SB-C18, specifications: 9.4 mm × 150 mm, 5 μm; acetonitrile-water = 65:35-75:25, v / v; flow rate: 4 mL / min; detection wavelength: 205 nm) gradient elution for 30 min to prepare 1.2 mg of compound 1 of the present invention (retention time 13.3 min).

[0045] Structural identification of the compound: The cytochalasin compound prepared in this example was dissolved in 0.5 mL of deuterated chloroform and transferred to an NMR tube using a 200 μL pipette. The proton, carbon, and two-dimensional spectra (e.g., 1H, 1C, and 2D spectra) were detected on an NMR spectrometer (Bruker Avance III 600 MHz). Figure 1-6 The structure of the compound was determined by combining various physicochemical data, and it was named boerelasin L.

[0046] The obtained NMR data of cytochalasin:

[0047]

[0048]

[0049] Other physicochemical data:

[0050] Appearance: Pale yellow oily substance; Specific rotation (c 0.05, MeOH); UV(MeOH)λ max (log ε)205(2.56)nm; High-resolution mass spectrometry HR-ESI-MS m / z 478.29513 [M+H] + (calcd for C 30 H 39 NO4, 478.29519).

[0051] Based on the results of the above tests, the structural formula of the compound obtained in this embodiment is confirmed as follows:

[0052]

[0053] Molecular formula: C 30 H 39 NO4

[0054] Example 2: CCK-8 assay for cell viability

[0055] Principle: The CCK-8 (Cell Counting Kit-8) reagent detects cell viability based on the principle of mitochondrial dehydrogenase-mediated biochemical reactions. A series of dehydrogenases in the mitochondria of living cells, such as succinate dehydrogenase and malate dehydrogenase, maintain stable activity during cellular metabolism. They can reduce exogenous water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt) to a highly water-soluble orange-yellow formazan product. With changes in cell viability, mitochondrial dehydrogenase activity is positively correlated with cell number and metabolic intensity. Higher cell viability and more vigorous metabolism result in a greater amount of dehydrogenase participating in the reaction, and a corresponding increase in the formazan product produced. The formazan product exhibits a characteristic absorption peak at 450 nm. The absorbance (OD value) at this wavelength, measured using a microplate reader, shows a strict linear relationship with the number of viable cells within a certain range. Since the CCK-8 reagent itself is non-cytotoxic and the generated formazan product is highly water-soluble, no additional procedures such as cell lysis or organic solvent extraction are required, allowing for direct detection. Therefore, this method enables real-time, dynamic monitoring of cell proliferation, toxicity, and survival status under different treatment conditions, providing a quantitative basis for assessing the effects of compounds on cell activity.

[0056] Experimental materials and instruments:

[0057] Cell lines: Mouse mononuclear macrophages (RAW 264.7, China Center for Type Culture Collection, Wuhan University); DMEM medium and sterile PBS (pH=7.4, 0.01M) (gibco); fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.); CCK-8 (Solarbio); DMSO (Biosharp); 96-well plates, cell scrapers, cell culture flasks (NEST); mouse TNF-α and IL-6 ELISA kits (Xinbosheng Biotechnology Co., Ltd.); COX-2, iNOS, GAPDH (Wuhan Aiboteke Biotechnology Co., Ltd.); HRP-goat anti-rabbit, SDS-PAGE protein loading buffer (5×), protease inhibitors, ECL chemiluminescence solution, RAPI lysis buffer, rapid blocking solution (Beyotime); multi-functional microplate reader (TECAN, Germany); CO2 incubator (Thermo); inverted microscope (Nikon); autoclave (Shanghai Boxun Medical Bio-Instrument Co., Ltd.); ice maker (Panasonic).

[0058] Experimental procedure: Take cells in good growth condition during the logarithmic growth phase, and use 1×10⁻⁶ cells... 5Cells were seeded per well into 96-well plates, with 100 μL of DMEM complete medium added to each well. The plates were then incubated in a constant temperature incubator (5% CO2, 37°C) for 12 h. The compound obtained in Example 1 was first dissolved in DMSO (10 mM), and then different concentrations of working solutions (1, 5, 10, 20, 30, 40 μM) were prepared using sterile, FBS-free DMEM medium for gradient assays. After cell attachment, the supernatant was discarded, and the cells were aspirated and then added to pre-prepared solutions (100 μL / well): Control group: DMEM medium (without fetal bovine serum); Model group: 1 μg / mL LPS solution; Drug group: ① 1 μg / mL LPS solution + 40 μM of the compound obtained in Example 1, ② 40 μM of the compound obtained in Example 1 (without LPS); each group had 3 replicates. The 96-well plate was incubated at 37°C with 5% CO2 for 24 hours. Then, 100 μL of the prepared CCK-8 detection solution (CCK-8:DMEM = 1:9, v / v) was added to each well. After incubation at 37°C with 5% CO2 for 1 hour, the plate was removed and the absorbance was measured at 450 nm using a microplate reader. The cell viability was calculated (drug group / blank group × 100%). A viability of ≥80% generally indicates that the compound has no obvious toxic effects.

[0059] Experimental Results: The cytotoxicity of this compound after co-culturing with RAW 264.7 macrophages was detected by the CCK-8 assay. The results showed that after incubation with 40 μM of the compound, and with or without LPS stimulation for 24 h, the cell viability was >80%, indicating that the compound had no significant cytotoxicity to either normal or LPS-induced RAW 264.7 macrophages.

[0060] Example 3: Anti-inflammatory activity assay

[0061] Principle: In the presence of H + In the desired environment, nitric oxide reacts with diazonium salt sulfanilamide to form a diazo compound. This diazo compound further couples with naphthylvinyldiamine to form a red compound, which exhibits a maximum absorption peak at 540-560 nm. Within an appropriate concentration range, the concentration of the red compound is linearly correlated with the absorbance value, and the concentration of the red compound formed in the reaction is linearly related to the concentration of nitric oxide. Therefore, the NO concentration can be estimated from the absorbance (OD value). Furthermore, the inhibitory effect of the compound obtained in Example 1 on the NO inflammatory effect induced by LPS in RAW 264.7 macrophages was investigated.

[0062] The principle of enzyme-linked immunosorbent assay (ELISA) for detecting IL-6 and TNF-α is based on the specific binding of antigen and antibody. In the lipopolysaccharide (LPS)-activated RAW 264.7 macrophage inflammatory cascade, IL-6 and TNF-α, as core pro-inflammatory cytokines, are mediated by the NF-κB signaling pathway, resulting in their transcriptional activation and massive secretion into the extracellular microenvironment. The ELISA kit is pre-coated with specific capture antibodies against IL-6 and TNF-α. When cell culture supernatant is added, IL-6 and TNF-α in the sample bind to the capture antibodies. The subsequently added enzyme-labeled detection antibody specifically recognizes the IL-6 and TNF-α already bound to the capture antibodies, forming a sandwich structure of "capture antibody-antigen-enzyme-labeled detection antibody". After the substrate is added, the enzyme label catalyzes a colorimetric reaction, and the amount of colored product generated is proportional to the concentration of IL-6 and TNF-α in the sample. By measuring the absorbance value at a specific wavelength using an ELISA reader and comparing it with a standard curve, the concentrations of IL-6 and TNF-α in the sample can be accurately calculated, thereby evaluating the inhibitory effect of the compound obtained in Example 1 on the secretion of inflammatory cytokines.

[0063] The principle of Western blotting (WB) for measuring COX-2 and iNOS expression is based on the specific recognition of proteins. COX-2 and iNOS are key proteins in the inflammatory response, and their expression levels are significantly upregulated in macrophages under LPS stimulation. In this experiment, total protein was first extracted from RAW 264.7 macrophages, and proteins of different molecular weights were separated by SDS-PAGE gel electrophoresis. Subsequently, the proteins in the gel were transferred to a solid membrane using a transfer technique. Specific primary antibodies against COX-2 and iNOS bound to the target proteins on the membrane, and then a secondary antibody labeled with horseradish peroxidase (HRP) was added. The secondary antibody specifically bound to the primary antibody to form an immune complex. After the addition of a chemiluminescent substrate, HRP catalyzed the substrate to emit light, and the luminescence intensity was detected using a chemiluminescence imaging system. The luminescence intensity was positively correlated with the expression levels of COX-2 and iNOS on the membrane. By analyzing the gray values ​​of the target protein bands and normalizing them with the internal reference protein, the regulatory effects of the compounds obtained in Example 1 on the expression of COX-2 and iNOS proteins can be accurately evaluated, revealing their anti-inflammatory mechanism at the protein level.

[0064] Experimental steps:

[0065] (1) Cell Culture: First, remove the frozen cells from the -80℃ freezer / liquid nitrogen. Using clean gloves or a float, place the cryovials in a 37℃ constant temperature water bath. After most of the cryopreservation solution (90% FBS + 10% DMSO) in the tubes has thawed, quickly transfer the cells to a sterile centrifuge tube, add an appropriate amount of solution (PBS buffer or DMEM medium; DMEM medium was used in this step), and centrifuge (1500 rpm, 5 min). Discard the supernatant and transfer the centrifuged cells to DMEM complete medium containing 10% FBS. Place the tubes in a 5% CO2 constant temperature incubator (37℃) and observe cell morphology and number every morning and evening. Once the cells have grown to a suitable number, pipette the cells and mix them thoroughly before passage.

[0066] (2) NO detection: Take cells in good growth state during the logarithmic growth phase and use 1×10⁻⁶ NO assay. 5 Cells were seeded per well into 96-well plates and cultured at 37°C with 5% CO2 for 12 hours. The culture medium was then aspirated slowly, taking care not to remove the cells. Control group: DMEM medium (without fetal bovine serum); Model group: 1 μg / mL LPS solution; Positive control group: 1 μg / mL LPS solution + 10 μM dexamethasone; Drug group 1: 1 μg / mL LPS solution + different concentrations of the compound obtained in Example 1 (1, 5, 10, 20, 30, 40 μM); Drug group 2: 1 μg / mL LPS solution + compound cytochalasin T (40 μM). 100 μL was injected into each well, with three replicates per group. The plates were incubated at 37°C with 5% CO2 for 24 hours. Take 50 μL of supernatant, and add 50 μL of solution A and solution B respectively according to the Griess method. Then, measure the absorbance at 540 nm using an ELISA reader. Calculate the NO concentration based on the OD value and the NO standard curve, and then determine the IC50 of the analyte. 50 NO inhibition rate = (model group - drug group) / (model group - blank group) × 100%.

[0067] cytochalasin T: A compound previously prepared in the inventor's laboratory, see "Lv Xiao, Ye Ke, Ma Xujun, et al. Study on chemical composition and anti-inflammatory activity of endophytic fungus Boeremia exigua from Fritillaria thunbergii in Hubei Province [J]. Journal of South-Central University for Nationalities (Natural Science Edition), 2022, 41(2): 174-179", the structural formula is as follows:

[0068]

[0069] (3) ELISA detection of pro-inflammatory factors IL-6 and TNF-α: Cells in good growth state during the logarithmic growth phase were used at a concentration of 1×10⁻⁶ cells. 5Cells were seeded per well into 96-well plates and cultured at 5% CO2 and 37°C for 12 hours, after which the culture medium was aspirated. Following step (2), blank group, model group, positive control group, and drug group were set up, with 6 replicates per group. After incubation at 5% CO2 and 37°C for 24 hours, the supernatant from the 6 replicates of the same group was collected (without aspirating the cells). The cells were then detected according to the instructions of the enzyme-linked immunosorbent assay kit (repeated 3 times). A standard curve was constructed, and the levels of relevant inflammatory factors were calculated.

[0070] (4) Western Blot assay for protein expression: Total cell protein extraction: Cells in good growth condition were subjected to a 2×10⁻⁶ saturation test. 5 Cells were seeded per well in 6-well plates, 2 mL per well. Incubated for 12 h at 37°C with 5% CO2, then the culture medium was aspirated. Following step (2), blank group, model group, positive control group, and drug group were set up, with two replicates per group. After culturing in an incubator for 24 h, the cells were removed and placed on ice. The supernatant was discarded. 1 mL of pre-chilled PBS (placed at 4°C) was added to each well, and the cells were gently washed once. Then, 1 mL of PBS was added to each well, and the cells were pipetted off the plate. All cells from each well were quickly collected on ice and placed in a 1.5 mL EP tube. The 1.5 mL EP tube was centrifuged at 4°C for 5 min (1500 rpm). The supernatant was aspirated as thoroughly as possible, being careful not to remove the cells. 200 μL of lysis buffer (basal lysis buffer: protease inhibitor = 50:1, v / v) was added to each tube, and the cells were lysed on ice for 30 min. After lysis, centrifuge at 4°C for 5 min (12000 rpm) and collect the supernatant (be careful not to collect the lower gelatinous layer). Collect the same group into the same 0.5 mL EP tube and then perform protein concentration determination: quantify the extracted protein according to the Beyotime kit instructions (BCA quantification method).

[0071] After the protein concentration was determined, 50 μL of loading buffer (SDS-PAGE protein loading buffer) was added to each group, shaken well, and then placed on a preheated 95℃ metal bath and boiled for 10 min to fully denature the protein (shaking well and preventing explosion during the process). The mixture was then aliquoted and stored in a -20℃ freezer.

[0072] Prepare PAGE gels using a one-step method: 0.75 / 1.0 / 1.5mm lower gel: 2.0 / 2.7 / 4.0 mL lower gel solution + 2.0 / 2.7 / 4.0 mL lower buffer + 40 / 60 / 80 μL modified coagulant; 0.75 / 1.0 / 1.5mm. Upper gel: 0.5 / 0.75 / 1.0 mL upper gel solution + 0.5 / 0.75 / 1.0 mL upper buffer + 10 / 15 / 20 μL modified coagulant. Add an equal amount of protein sample to the electrophoresis wells. Upper layer separation conditions: 90V constant voltage for 30 min; lower layer separation conditions: 140V constant voltage for 60 min. Cut an appropriate size of PVDF membrane, activate it first in chromatographic methanol, then remove the separating gel, arrange it in order, paying attention to the positive and negative electrodes, and transfer the membrane at a constant current of 200 mA for 2 h. TBST preparation: 2.4g Tris, 8g NaCl, and ultrapure water were added to a final volume of 1L. After transfer, rapid blocking buffer was added (blocking at room temperature for 30 min). After blocking, the membrane was washed four times with TBST for 5 min each time, and then incubated with primary antibody at 4°C for 12 h. The PVDF membrane incubated with primary antibody was removed from the refrigerator, washed four times with TBST for 5 min each time, and then incubated with secondary antibody at room temperature for 2 h. After two incubations and washing with TBST, freshly prepared ECL chemiluminescence solution was pipetted onto the PVDF membrane, and finally, the membrane was placed on a gel imaging system for development. The same procedure was repeated three times for protein electrophoresis.

[0073] Experimental results:

[0074] Anti-inflammatory results such as Figure 7 As shown, treatment with different concentrations of the compound significantly inhibited NO production in a dose-dependent manner, with an IC50 value of [missing information]. 50 The concentration was 8.56 μM, and the NO production inhibition rate was 91.86% at a concentration of 40 μM. The results indicate that this compound has a low-toxicity and highly effective inhibitory effect on NO production by LPS-stimulated RAW 264.7 macrophages, while cytochalasin T showed a NO inhibition rate of <10% at 40 μM and had no anti-inflammatory activity.

[0075] The collected cell supernatant was analyzed using an ELISA kit, and the results are as follows: Figure 8 As shown: Compared to the control group, LPS stimulation increased IL-6 concentration by nearly 8 times. ###The compound (P<0.001) increased the concentration of TNF-α by nearly 20 times. Different concentrations of the compound (5, 10, 20 μM) significantly reduced the expression of IL-6 and TNF-α inflammatory factors. The compound-treated groups showed significant differences compared to the LPS group (**P<0.01 and ***P<0.001). Furthermore, compared to the DEX group (positive control dexamethasone 10 μM), when the concentration of compound 1 was >10 μM, the compound's anti-inflammatory effect was significantly better than the positive control, indicating that the compound can inhibit the expression of inflammatory factors and has an anti-inflammatory effect.

[0076] like Figure 9 The expression of LPS-induced inflammation-related factors COX-2 and iNOS was detected by the compound. Western blot analysis showed that the LPS group significantly activated the expression of COX-2 and iNOS compared to the Control group. ### (P<0.001) The compound at a dose of 5 μM was able to significantly inhibit the expression of inflammatory factors, and the downregulation effect on iNOS was more obvious. Compared with the DEX group (10 μM), when the compound concentration was ≥10 μM, the inhibitory effect of compound 1 was more obvious, and it had a dose-dependent downregulation effect on the expression of COX-2 and iNOS proteins.

Claims

1. A novel cytochalasin-like compound with a novel skeletal structure, the structural formula of which is:

2. A method for preparing the cytochalasin compound of claim 1, comprising the following steps: The fermentation product of the endophytic fungus Boeremia exigua from Fritillaria hupehensis on oat solid medium was extracted by soaking in a chloroform-methanol mixture. The extract was concentrated to remove the solvent, then dissolved in water and extracted with ethyl acetate. The ethyl acetate fraction was concentrated to obtain an extract. The extract was then purified by normal-phase silica gel column chromatography, medium-pressure liquid chromatography, Sephadex LH-20 gel column chromatography, normal-phase silica gel column chromatography, and high-performance liquid chromatography to obtain the cytochalasin-like compounds.

3. The use of the cytochalasin-like compound of claim 1 in the preparation of a drug for inhibiting NO production.

4. The use of the cytochalasin compound of claim 1 in the preparation of a drug that downregulates the expression levels of iNOS and / or COX-2.

5. The use of the cytochalasin compound of claim 1 in the preparation of a drug that inhibits the secretion of inflammatory factors IL-6 and / or TNF-α.

6. The use of the cytochalasin compound of claim 1 in the preparation of anti-inflammatory drugs.

7. A medicament for treating metabolic, immune regulation, and / or signal transduction-related diseases caused by excessive levels of NO at physiological levels, characterized in that, The drug uses the cytochalasin compound of claim 1 as its main active ingredient.

8. The medicament according to claim 7, characterized in that, The drug uses the cytochalasin compound of claim 1 as its sole active ingredient.

9. An anti-inflammatory drug, characterized in that, The drug uses the cytochalasin compound of claim 1 as its main active ingredient.

10. The anti-inflammatory drug according to claim 9, characterized in that, The drug uses the cytochalasin compound of claim 1 as its sole active ingredient.

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