A heterozygous sesquiterpene tricyclic epoxy compound derived from a oligosporous Aranea sp. strain, its preparation method and application

Abstract

This invention discloses a heterozygous sesquiterpene tricyclic epoxy compound derived from the strain *Arthrum oligosporum*, its preparation method, and its applications, belonging to the field of natural product chemistry. This invention provides a heterozygous sesquiterpene tricyclic epoxy compound derived from *Arthrum oligosporum* (… A.oligospora ) AOL_ s00210g57 Anthrobotrisin D, a novel heterozygous sesquiterpene tricyclic epoxy compound from a gene knockout strain, has been shown in vitro to possess excellent anti-inflammatory activity, significantly inhibiting lipopolysaccharide-induced inflammatory responses in mouse microglia. It exhibits a clear dose-dependent inhibitory trend at multiple levels, including NO release, protein and mRNA expression of inflammatory factors (TNF-α, IL-1β, IL-6), and iNOS protein and mRNA expression. Furthermore, at a concentration of 33 μM, its anti-inflammatory effect is comparable to that of the clinically proven positive drug dexamethasone, and in some aspects, it is even superior. This invention not only provides a new lead compound for the development of anti-inflammatory drugs but also opens up new directions for developing new drugs from the unique microbial resource of nematode-preying fungi.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to a heterozygous sesquiterpene tricyclic epoxy compound derived from the Oligosporium nobile strain, its preparation method, and its application. Background Technology

[0002] Microorganisms are an important resource for new drug development, and fungi, with their diverse metabolic pathways and abundant secondary metabolites, have long been considered a core source for drug screening. Fungal metabolites exhibit a wide range of physiological activities, encompassing antibacterial, antitumor, immunomodulatory, anti-inflammatory, and enzyme-inhibiting functions, playing a crucial role in clinical drug development. Currently, mining novel drug molecules from fungi has become a research hotspot in the international and domestic biopharmaceutical fields. However, most biosynthetic gene clusters are silent under laboratory conditions, and only a small portion of these clusters are transcribed. To expand metabolite diversity, researchers have employed various strategies, including epigenetic modification and genome mining, to activate silent gene clusters.

[0003] Inflammation, as an important immune response of the body to exogenous infection, is closely related to various diseases such as Alzheimer's disease, obesity, diabetes, atherosclerosis, and cancer when out of control. Currently, the anti-inflammatory drugs commonly used in clinical practice are mainly divided into nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SSAIDs). Although these two types of drugs can relieve inflammatory symptoms in the short term, long-term and high-dose use can easily cause adverse reactions such as gastric mucosal damage, abnormal liver function, and kidney damage. In addition, some patients will develop drug tolerance. There is an urgent need to develop new anti-inflammatory active molecules with higher safety and better efficacy.

[0004] To address the aforementioned problems, this invention aims to provide a compound with excellent anti-inflammatory activity derived from a genetically engineered mutant strain of Oligosporium nobile, providing key strain resources and molecular basis for the development of novel anti-inflammatory drugs, thereby solving the problem that long-term use of existing anti-inflammatory drugs easily causes adverse reactions such as gastric mucosal damage and liver damage. Summary of the Invention

[0005] The first objective of this invention is to provide a heterozygous sesquiterpene tricyclic epoxy compound. The second objective of this invention is to provide a method for preparing the compound and its applications.

[0006] The first objective of this invention is achieved by providing a heterocyclic sesquiterpene tricyclic epoxy compound with the structural formula shown in Formula I:

[0007] .

[0008] The heterozygous sesquiterpene tricyclic epoxy compound was isolated from Oligosporium. (A. oligospora) strain YMF1.3170 AOL_s00210g57 Gene knockout strain.

[0009] The second objective of this invention is achieved by the method for preparing the heterocyclic sesquiterpene tricyclic epoxy compound, which is carried out according to the following steps:

[0010] 1) AOL_s00210g57 After the gene knockout strain was activated and cultured in seed culture medium, it was inoculated into fermentation culture medium and fermented for 3-7 days to obtain fermentation broth.

[0011] 2) Crude extraction: The obtained fermentation broth is concentrated to a certain volume under reduced pressure and extracted with an equal amount of ethyl acetate. The extract is then concentrated to obtain an extract.

[0012] 3) Separation and purification: The obtained extract was first separated by reversed-phase RP-18 column chromatography using a gradient elution with methanol-water solutions of volume ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. Fraction Fr. J-5 was then purified by semi-preparative high-performance liquid chromatography using acetonitrile-water solutions of a constant volume ratio of 34:66. The fraction with a retention time of 17.9 minutes was collected.

[0013] 4) The obtained fraction was further separated by silica gel column chromatography, and gradient elution was performed with petroleum ether-acetone solutions at volume ratios of 4:1, 3:1, and 2:1 to obtain the target heterocyclic sesquiterpene tricyclic epoxy compound.

[0014] The application of the hetero-sesquiterpene terpene epoxy compound is in the preparation of an anti-inflammatory drug that can inhibit LPS-induced NO production.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention provides a method for obtaining oligosporous arbuscular mycorrhizal (Syndromea spp.) A. oligospora ) AOL_s00210g57Anthrobotrisin D, a novel heterozygous sesquiterpene tricyclic epoxy compound from a gene knockout strain, has been shown in vitro to possess excellent anti-inflammatory activity and low cytotoxicity. It significantly inhibits lipopolysaccharide (LPS)-induced inflammatory responses in mouse microglia (BV2 cells). It exhibits a clear dose-dependent inhibitory trend at multiple levels, including NO release, protein and mRNA expression of inflammatory factors (TNF-α, IL-1β, IL-6), and iNOS protein and mRNA expression. At a concentration of 33 μM, its anti-inflammatory effect is comparable to that of the clinical positive control drug dexamethasone, and in some aspects, it is even superior. Furthermore, MTT assay results show that anthrobotrisin D inhibits BV2 cells by less than 1% within the concentration range of 3.3–100 μM, maintaining cell viability above 99% with no significant cytotoxicity.

[0017] This invention is the first to discover a novel heterozygous sesquiterpene derivative with significant anti-inflammatory activity and good safety from this gene knockout strain. It not only provides a new lead compound for the development of anti-inflammatory drugs, but also opens up a new direction for the development of new drugs from the unique microbial resource of nematode-preying fungi. It is of great significance for solving the adverse reaction problem of existing anti-inflammatory drugs and promoting the development of treatment technology for inflammatory diseases. Attached Figure Description

[0018] Figure 1 shows the chemical structural formula of compound I of the present invention;

[0019] Figure 2 shows the mass spectrum of compound G57-6 of this invention;

[0020] Figure 3 is the proton spectrum of compound G57-6 of the present invention;

[0021] Figure 4 shows the carbon spectrum of compound G57-6 of this invention;

[0022] Figure 5 shows the HMBC spectrum of compound G57-6 of this invention;

[0023] Figure 6 shows the COSY spectrum of compound G57-6 of this invention;

[0024] Figure 7 shows the HSQC spectrum of compound G57-6 of this invention;

[0025] Figure 8 shows the rotation spectrum of compound G57-6 of this invention;

[0026] Figure 9 shows the effects of compound G57-6 of the present invention on LPS-induced NO release and iNOS expression in BV2 cells: A: Effect of G57-6 on LPS-induced nitrite content in BV2 cells; B: Effect of G57-6 on LPS-induced iNOS expression in BV2 cells.

[0027] Figure 10 shows the effects of compound G57-6 of the present invention on LPS-induced inflammatory factors TNF-α, IL-1β, and IL-6 in BV2 cells: A: TNF-α level; B: IL-1β level; C: IL-6 level; D: TNF-α mRNA expression level; E: IL-1β mRNA expression level; F: IL-6 mRNA expression level.

[0028] Figure 11 shows the effect of compound G57-6 of the present invention on the expression of the inflammatory mediator iNOS protein induced by LPS in BV2 cells: A: iNOS protein band; B: gray value analysis of iNOS / GAPDH protein band. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0030] This invention provides a heterocyclic sesquiterpene tricyclic epoxy compound, the structural formula of which is shown in Formula I:

[0031] .

[0032] The heterozygous sesquiterpene tricyclic epoxy compound was isolated from the strain A. oligospora YMF1.3170. AOL_s00210g57 Gene knockout strain.

[0033] The present invention also provides a method for preparing the aforementioned hetero-sesquiterpene tricyclic epoxy compound, which is carried out according to the following steps:

[0034] 1) AOL_s00210g57 After the gene knockout strain was activated and cultured in seed culture medium, it was inoculated into fermentation culture medium and fermented for 3-7 days to obtain fermentation broth.

[0035] 2) Crude extraction: The obtained fermentation broth is concentrated to a certain volume under reduced pressure and extracted with an equal amount of ethyl acetate. The extract is then concentrated to obtain an extract.

[0036] 3) Separation and purification: The obtained extract was first separated by reversed-phase RP-18 column chromatography using a gradient elution with methanol-water solutions of volume ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. Fraction Fr. J-5 was then purified by semi-preparative high-performance liquid chromatography using acetonitrile-water solutions of a constant volume ratio of 34:66. The fraction with a retention time of 17.9 minutes was collected.

[0037] 4) The obtained fraction was further separated by silica gel column chromatography, and gradient elution was performed with petroleum ether-acetone solutions at volume ratios of 4:1, 3:1, and 2:1 to obtain the target heterocyclic sesquiterpene tricyclic epoxy compound.

[0038] In step 1), the seed culture medium is PDA medium, which consists of: 200 g / L potato, 20 g / L glucose, 15-20 g / L agar, water as solvent, and natural pH.

[0039] In step 1), the activation culture conditions are 7 days of culture at 25 ℃.

[0040] In step 1), the fermentation medium is PDB fermentation medium, and the fermentation conditions are 25 ℃, 180 rpm / min shaker culture for 7 days.

[0041] The present invention also provides the application of the aforementioned hetero-sesquiterpene terpene epoxy compound in the preparation of anti-inflammatory drugs.

[0042] The anti-inflammatory drug can inhibit LPS-induced NO production.

[0043] The present invention relates to oligosporous arbuscular mycorrhizal (… A. oligospora ) AOL_s00210g57 The gene knockout strain was constructed using the wild-type strain YMF 1.3170 of Oligosporium nobile as the starting strain; AOL_s00210g57 The relevant background information of the gene knockout strain is as follows: This strain comes from the State Key Laboratory of Yunnan Provincial Biological Resources Conservation and Utilization, which is jointly established by Yunnan University and Yunnan Province. For details, please refer to the literature "Preliminary Study on the Omics of Five Biosynthetic Genes in Oligosporium and Fungal Morphology and Secondary Metabolites" (Yunnan University, 2016). The strain was obtained from Researcher Niu Xuemei of Yunnan University.

[0044] Example 1: Preparation and identification of anthrobotrisin D, a heterozygous sesquiterpene terpene epoxide compound

[0045] This embodiment utilizes Oligosporium argentis (… A. oligospora strain YMF 1.3170 AOL_s00210g57 Anthrobotrisin D, a heterozygous sesquiterpene tricyclic epoxidized compound, was produced by fermentation using a gene knockout strain. The specific experimental methods and results are as follows:

[0046] 1. AOL_s00210g57 The gene knockout strain was inoculated into PDA medium and cultured at 25°C for 7 days. The components of the medium (by weight ratio) were: 200 g potato, 20 g glucose, 15-20 g agar, 1000 mL water, and natural pH.

[0047] 2. For large-scale liquid fermentation, 60 L of seed culture was inoculated into PDB medium (121 ℃, sterilized for 30 min, pH 7.2), and cultured at 25 ℃ and 180 r / min for 7 days.

[0048] 3. The fermentation broth was concentrated to 3 L using a rotary evaporator, and extracted three times with an equal volume of ethyl acetate. The ethyl acetate fraction was then concentrated to obtain the extract.

[0049] 4. 1.2 g of the ethyl acetate extract was dissolved in methanol, filtered, and separated by reversed-phase RP-18 column chromatography (methanol:water, 10:100→100:0) to obtain fractions Fr. A to Fr. M. Fraction Fr. J-5 was isocratically eluted by semi-preparative HPLC (acetonitrile:water = 34:66, tR3 = 17.9 min), followed by silica gel column chromatography (petroleum ether:acetone, 4:1→2:1) to obtain compound G57-6 (8.2 mg).

[0050] The structure of compound G57-6 was determined, and its physicochemical data are as follows: -13.64 ( c 0.11,MeOH); HR-ESI-MS m / z: 415.2087 [M+Na] + (calcd. for C 22 H 32 Compound G57-6 is a colorless, oily liquid. Its molecular formula was determined to be C6 by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) in positive ion mode. 22 H 32 O6 corresponds to a high-resolution mass spectrometry value of m / z 415.2091 [M+Na]. +(Sodium adduct ion peak), theoretically calculated value 415.2091, indicating that the degree of unsaturation of this compound is 7. The ¹H NMR spectrum of compound G57-6 in methanol (Table 1) shows four methyl hydrogen signals at chemical shifts δH 1.81, 1.69, 1.62, and 1.61, and three olefin hydrogen signals at δH 5.99, 5.30, and 5.00; its 13 The C NMR spectrum (Table 1) shows a total of 22 carbon signals, including 4 methyl carbons, 4 methylene carbons (1 of which is an oxygen-containing compound), and 8 methine carbons (4 of which are oxygen-containing compounds and 4 are sp). 2 Hybridized carbons) and 6 hydrogen-free carbons (one of which is a carbonyl carbon); in 1 H- 1 In the HCOSY spectrum, correlations exist between 5.05 and 5.30, 3.92 and 2.23, 2.23 and 5.00, 1.75 and 2.05, and 2.05 and 5.10. In the HSQC spectrum, correlations exist between 66.7 and 4.67, 120.6 and 5.99, 58.4 and 3.86, 62.3 and 4.28, 4.40, 63.9 and 5.05, 124.7 and 5.30, 78.4 and 3.92, 34.8 and 2.23, 121.5 and 5.00, 41.0 and 1.95, 27.9 and 2.05, 125.6 and 5.10, 26.0 and 1.69, 17.9 and 1.62, 16.5 and 1.61, and 12.4 and 1.81. In summary, this compound was identified as a novel heterozygous sesquiterpene tricyclic epoxy group and named anthrobotrisin D.

[0051] Table 1. Compound G57-6 1 H and 13 C NMR data (acetone-d6)

[0052]

[0053] Example 2: Detection of cytotoxicity and anti-inflammatory activity of the heterozygous sesquiterpene tricyclic epoxy compound G57-6

[0054] I. Cell Culture and Compound Preparation

[0055] Mouse BV2 cells were seeded in DMEM high-glucose medium containing 10% FBS and cultured and passaged routinely at 37°C with 5% CO2 saturated humidity. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were selected for subsequent experiments. Lipopolysaccharide (LPS) used in this experiment was prepared by dissolving it in sterile PBS. Compounds G57-6 and dexamethasone (DXM) were dissolved in cell-grade DMSO and serially diluted to prepare stock solutions of various concentrations (ensuring the final DMSO concentration was ≤1% to avoid solvent toxicity).

[0056] II. Cytotoxicity and Anti-inflammatory Activity Detection

[0057] 1. Cytotoxicity detection

[0058] This experiment used the MTT assay to detect the toxicity of compound G57-6 to BV2 cells in order to avoid false positive results caused by interference from cytotoxicity.

[0059] Digest healthy BV2 cells to prepare a cell suspension (viable cell rate ≥95%), and adjust the cell concentration to 1×10⁻⁶. 5 Cells per well were seeded in 96-well plates and incubated overnight at 37°C with 5% CO2 to achieve cell adhesion of over 80%. Two groups were set up: a blank control group and compound G57-6 treatment groups (final concentrations of 3.3 μM, 11 μM, 33 μM, and 100 μM, respectively), with three replicates per group. The next day, 1 μL of G57-6 stock solution dissolved in DMSO was added to each well of the compound G57-6 treatment group, while the blank control group was treated with an equal volume of DMSO. Both groups were incubated for 24 hours, followed by 20 μL of 5 mg / mL MTT solution and incubation for another 4 hours. The culture medium was then discarded, and 150 μL of DMSO was added. The plates were shaken at low speed for 10 minutes to fully dissolve the purple crystals. The inhibition rate was calculated by measuring the OD value at 490 nm using a microplate reader.

[0060] Results: As shown in Table 1, compound G57-6 inhibited BV2 cells by less than 1% in the concentration range of 3.3 to 100 μM, indicating that compound G57-6 had no significant toxicity to BV2 cells in this concentration range.

[0061] Table 2 Effects of compounds on BV2 cell survival

[0062]

[0063] 2. Anti-inflammatory activity assay

[0064] (1) Effect of the compound on LPS-induced NO release from BV2 cells

[0065] BV2 cells in the logarithmic growth phase were digested and prepared into a cell suspension, then diluted with 1×10⁻⁶ cells. 6 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated overnight in a 5% CO2, 37°C incubator to achieve cell adhesion of over 80%. Four experimental groups were set up: a blank control group, an LPS model group (1 μg / mL LPS), a positive control group (1 μg / mL LPS + 33 μM DXM), and compound G57-6 treatment groups (1 μg / mL LPS + 3.3 μM, 1 μg / mL LPS + 11 μM, 1 μg / mL LPS + 33 μM). The specific procedures were as follows: The next day, the medium was replaced with 1 mL of serum-free DMEM. 1 μl of DMSO was added to the blank control group and the LPS model group, while the other groups were pretreated with equal volumes of compound G57-6 (final concentrations of 3.3 μM, 11 μM, and 33 μM) or dexamethasone (final concentration of 33 μM). After 2 hours of pretreatment, except for the blank control group, all other groups were stimulated with LPS (final concentration of 1 μg / mL) for 24 hours. After incubation, the concentration of nitrite in the supernatant was determined using the Griess method to quantify NO levels. Griess Reagent I and II were brought to room temperature, and standards were diluted with serum-free DMEM medium to concentrations of 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM. 50 μL of each sample and standard was added to a new 96-well plate, with three replicates per sample. Then, 50 μL of Griess Reagent I was added to each well, followed by Griess Reagent II. The absorbance was measured at 540 nm.

[0066] The results, as shown in Figure 9A, indicate that the nitrite (NO metabolite) content in BV2 cells decreased in a dose-dependent manner with increasing G57-6 concentration, suggesting that compound G57-6 can dose-dependently inhibit LPS-induced accumulation of NO metabolites in BV2 cells. At a concentration of 33 μM, its inhibitory effect was comparable to that of the positive control drug dexamethasone at the same concentration, indicating that G57-6 can effectively inhibit LPS-induced NO release, and no cytotoxicity was observed within this concentration range, preliminarily suggesting that compound G57-6 possesses good anti-inflammatory activity.

[0067] (2) ELISA method was used to detect the levels of inflammatory factors TNF-α, IL-1β and IL-6.

[0068] BV2 cells in the logarithmic growth phase were digested and prepared into a cell suspension, then diluted with 1×10⁻⁶ cells. 6 BV2 cells were seeded at a density of 100 cells / well in 6-well plates and incubated overnight at 37°C with 5% CO2 until cell adhesion reached over 80%. BV2 cells in the logarithmic growth phase were then seeded at a density of 1 × 10⁻⁶ cells / well.6 Cells were seeded at a density of 100 cells / well in 6-well cell culture plates and incubated overnight in a 5% CO2, 37°C incubator to achieve a cell adhesion rate of over 80%. Four experimental groups were set up: a blank control group, an LPS model group (1 μg / mL LPS), a positive control group (1 μg / mL LPS + 33 μM DXM), and compound G57-6 treatment groups (1 μg / mL LPS 3.3 μM, 1 μg / mL LPS + 11 μM, 1 μg / mL LPS + 33 μM). The following day, 1 mL of serum-free DMEM medium was replaced in each well. Except for the blank control group and the LPS model, 1 μl of DMSO was added to each of the other groups. The remaining groups were pretreated with equal volumes of compound G57-6 (final concentrations of 3.3 μM, 11 μM, and 33 μM) or dexamethasone (final concentration of 33 μM) for 2 hours. After 24 hours of co-stimulation with LPS (final concentration of 1 μg / mL) except for the blank control group, the cell supernatant was collected and the levels of inflammatory factors TNF-α, IL-1β, and IL-6 were detected according to the instructions of the mouse TNF-α, IL-1β, and IL-6 ELISA kit.

[0069] Results analysis: As shown in Figure 10 (AC), compared with the LPS model group, the levels of the three inflammatory factors in each G57-6 concentration treatment group were significantly reduced in a dose-dependent manner; at a concentration of 33 μM, the inhibitory effect of G57-6 on TNF-α, IL-1β, and IL-6 was comparable to that of the positive drug dexamethasone group, further confirming its strong anti-inflammatory activity.

[0070] (3) RT-qPCR detection of the expression of various inflammatory factors in BV2 cells

[0071] BV2 cells in the logarithmic growth phase were digested and prepared into a cell suspension, then diluted with 1×10⁻⁶ cells. 6Cells were seeded at a density of 100 cells / well in 6-well plates and incubated overnight in a 5% CO2, 37°C incubator to achieve cell adhesion of over 80%. Four experimental groups were set up: a blank control group, an LPS model group (1 μg / mL LPS), a positive control group (1 μg / mL LPS + 33 μM DXM), and compound G57-6 treatment groups (1 μg / mL LPS 3.3 μM, 1 μg / mL LPS + 11 μM, 1 μg / mL LPS + 33 μM). The following day, 1 mL of serum-free DMEM medium was added to each well. 1 μL of DMSO was added to the blank control group and the LPS model, while the remaining groups were pretreated for 2 hours with equal volumes of compound G57-6 (final concentrations of 3.3 μM, 11 μM, and 33 μM) or dexamethasone (final concentration of 33 μM). After this 2-hour pretreatment, all groups except the blank control group were stimulated with LPS (final concentration of 1 μg / mL) for 24 hours. After culture, cells were collected, washed three times with PBS, and the PBS residue was aspirated. 500 μL of Trizol reagent was added to each well to extract RNA, and the RNA content and purity were measured. Then, the RNA was reverse transcribed into cDNA using a reverse transcription kit. Using this cDNA as a template, quantitative real-time PCR was performed with primers for TNF-α, IL-1β, IL-6, iNOS, and the internal control (GAPDH) to detect the levels of inflammatory factors TNF-α, IL-1β, IL-6, and iNOS. Figure 10 DF) and iNOS ( Figure 9 The expression of B).

[0072] Results analysis: such as Figure 9 As shown in B, the relative expression level of iNOS mRNA decreased in a dose-dependent manner with increasing G57-6 concentration. The iNOS mRNA expression level in the 33 μM concentration group was comparable to that in the positive control group (dexamethasone), further demonstrating that G57-6 can reduce NO release by inhibiting iNOS gene transcription and decreasing NO synthase production, consistent with the results observed in step (1).

[0073] As shown in Figure 10 (DF), compared with the LPS model group, the expression levels of the three inflammatory factors mRNAs in the treatment groups with different concentrations of G57-6 were significantly reduced in a dose-dependent manner. This is consistent with the results of the extracellular ELISA method detected in step (2) above, proving that compound G57-6 downregulates the expression levels of these three inflammatory factors mRNAs. TNF-α , IL-1β and IL-6 It reduces the gene transcription level. It decreases the release of these extracellular inflammatory factors, thereby exerting an anti-inflammatory effect.

[0074] (4) Immunoblotting analysis of iNOS expression

[0075] BV2 cells in the logarithmic growth phase were digested and prepared into a cell suspension, then diluted with 1×10⁻⁶ cells. 6 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated overnight in a 5% CO2, 37°C incubator to achieve cell adhesion of over 80%. Four experimental groups were set up: a blank control group, an LPS model group (1 μg / mL LPS), a positive control group (1 μg / mL LPS + 33 μM DXM), and compound G57-6 treatment groups (1 μg / mL LPS 3.3 μM, 1 μg / mL LPS + 11 μM, 1 μg / mL LPS + 33 μM). The following day, 1 mL of serum-free DMEM medium was replaced in each well. 1 μL of DMSO was added to the blank control group and the LPS model, while the remaining groups were pretreated for 2 hours with equal volumes of compound G57-6 (final concentrations of 3.3 μM, 11 μM, and 33 μM) or dexamethasone (final concentration of 33 μM), respectively. After this 2-hour pretreatment, all groups except the blank control group were stimulated with LPS (final concentration of 1 μg / mL) for 24 hours. After stimulation, cells were collected, washed three times with pre-cooled PBS, and all residual liquid was thoroughly aspirated. Cells were then lysed to extract total protein, and protein concentration was determined using the BCA method. An equal volume of total protein (40 μg) was separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with TBS solution containing 5% skim milk at room temperature for 1 hour, followed by overnight incubation with the corresponding primary antibody at 4°C. After washing three times with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour, and then washed three more times with TBST. Finally, chemiluminescent substrates were used for imaging and detection on the FluorChem FC3 imaging system. GAPDH was used as an internal control, and the grayscale values ​​of the target bands were analyzed using the system's accompanying software.

[0076] Results Analysis: Western blot analysis (Figure 11A) showed that the iNOS protein expression level in the LPS model group was significantly increased compared with the blank control group; while treatment with different concentrations (3, 11, 33 μM) of G57-6 inhibited iNOS protein expression in a dose-dependent manner compared with the LPS model group. Statistical analysis of protein band gray values ​​(…) Figure 11 B) This result was further confirmed. At the protein level, this result further confirmed that compound G57-6 can reduce NO release by inhibiting iNOS protein expression and decreasing NO synthase production, exhibiting a good dose-dependent effect. In summary, this demonstrates that compound G57-6 exerts its anti-inflammatory effect by inhibiting NO release through the suppression of iNOS expression.

[0077] All data were processed and analyzed using SPSS 13.0 software. Measurement results are expressed as mean ± standard error (X ± SEM). After a second test of homogeneity of variance, the t-test was used to compare the two groups. p < 0.05 indicated a significant difference.

Claims

1. The application of a heterocyclic sesquiterpene terpene epoxy compound in the preparation of anti-inflammatory drugs, characterized in that, The structural formula of the hetero-sesquiterpene tricyclic epoxy compound is shown in Formula I: ； The heterozygous sesquiterpene terpene epoxy compounds all showed a clear dose-dependent inhibitory trend on NO release and the protein and mRNA expression of inflammatory factors TNF-α, IL-1β, IL-6, and iNOS.

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