Cyclohexenone compound, preparation method and application thereof

Abstract

The application discloses a cyclohexenone compound and a preparation method and application thereof, the compound has a structure shown in formula 1, and is obtained by being separated and purified from a fermentation product of a deep soil source fungus. The compound is obtained by the following steps: adopting a deep soil fungus Sordariomycetes sp. GZWMJZ-1598, using a solid state fermentation mode, after fermentation, and then through extraction, monomer separation and purification. The structure of the compound is characterized and confirmed by high resolution mass spectrometry, nuclear magnetic resonance spectroscopy, single crystal X diffraction and other methods. The cyclohexenone compound has a significant inhibitory effect on the NO release amount of LPS-induced RAW264.7 inflammatory cells, and can be applied to preparation of anti-inflammatory drug preparations.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a cyclohexenone compound and a preparation method and application thereof. BACKGROUND

[0002] Cyclohexenone is an important organic intermediate and is widely used in the fields of medicine, pesticide and the like. In a natural environment, a biologically synthesized multi-site substituted cyclohexenone derivative has the characteristics of green and low toxicity. The research on secondary metabolites of soil fungi is an important way for the development of natural products. Deep soil fungi have evolved special metabolic pathways to adapt to the extreme environment of high pressure, low oxygen, no light and oligotrophic habitat, thereby producing rich secondary metabolites with novel structures and excellent activities. The focus of the patent is that the inventors obtained a novel cyclohexenone compound through isolation and identification in the continuous research on deep soil fungi Sordariomycetes sp.GZWMJZ-1598, and the cyclohexenone compound has a significant inhibitory effect on the NO release amount of LPS-induced RAW264.7 inflammatory cells. NO is released by inflammatory cells such as macrophages in the inflammatory process, can induce the production of inflammatory cytokines, and aggravate the inflammatory response. By inhibiting the release of NO, the release of inflammatory mediators can be reduced, thereby reducing the symptoms of inflammation. Therefore, the discovery of the NO release amount inhibitory active molecule has important significance for anti-inflammatory diseases. SUMMARY

[0003] The purpose of the application is to provide a cyclohexenone compound, a preparation method and application thereof, which are obtained by extraction, separation and purification from the fermentation products of deep soil fungi, have an anti-inflammatory effect, and can be applied in anti-inflammatory drugs.

[0004] The above-mentioned purpose is achieved by the following technical solutions.

[0005] A cyclohexenone compound, which has the structure shown in formula 1:

[0006]

[0007] The compound 1 is obtained by separation and purification from the fermentation products of deep soil fungi, wherein the deep soil fungi are named Sordariomycetes sp.GZWMJZ-1598, and the preservation unit is preserved in the China Center for Type Culture Collection, Wuhan, China, on June 06, 2025, and the preservation number is CCTCC NO:M 20251925.

[0008] The preparation method of the compound 1 comprises the following steps:

[0009] A strain preparation: the weight ratio of potato, glucose and agar used in the purification medium is 200-300, 20-25 and 15-20 respectively, which is added to 1 L of pure water, the pH value is adjusted to 6.5-7.5, and then the medium is made into an inclined plane and placed at room temperature, and then the mycelium of Sordariomycetes sp. GZWMJZ-1598 is inoculated and cultured at 25-32℃ for 3 days as a strain;

[0010] B inoculum preparation: the volume ratio of potato and glucose used in the inoculum medium is 200-300 and 20-25 respectively, which is added to 1 L of pure water, the pH value is adjusted to 6.5-7.5, and then the medium is made into a liquid medium and placed at room temperature, and then the strain obtained in step A is inoculated and cultured at 25-32℃ with a rotation speed of 150-200 r / min for 3 days as an inoculum;

[0011] C inoculation: the solid fermentation method is used, 30-100 parts of rice and 30-100 parts of pure water are added to each fermentation bag, and the inoculum obtained in step B is inoculated and cultured at 28℃ for 30 days to obtain a fermentation medium;

[0012] D extraction: the fermentation medium described in step C is soaked with ethyl acetate for 48-72 hours, and then stirred with a stirrer for 3-5 times, each time for 30-40 minutes, and then the supernatant is combined and the ethyl acetate is recovered on a rotary evaporator to obtain a crude extract;

[0013] E monomer separation and purification: the crude extract obtained in step D is separated by column chromatography to obtain compound 1; the column chromatography method includes silica gel column chromatography, gel column chromatography and semi-preparative high performance liquid chromatography.

[0014] The cyclohexenone compound is used in the preparation of an anti-inflammatory drug.

[0015] A pharmaceutical composition comprising a cyclohexenone compound having the structural formula 1 and a pharmaceutically acceptable excipient.

[0016] The pharmaceutical composition contains 0.1-99% of the cyclohexenone compound having the structural formula 1 by mass fraction, and the rest is a pharmaceutical carrier or excipient.

[0017] The inventors have conducted more systematic research on the extraction, separation and purification of the fermentation product of the deep soil fungus Sordariomycetes sp. GZWMJZ-1598 in the continuous research on the fungus, and obtained a novel cyclohexenone compound 1. The prepared cyclohexenone compound has an inhibitory effect on the NO release amount of LPS-induced RAW264.7 inflammatory cells, and can be applied in anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a structural formula of compound 1 of the present application

[0019] Figure 2 is a single crystal graph of compound 1 of the present application

[0020] Figure 3 is a high resolution mass spectrum graph of compound 1 of the present application

[0021] Figure 4 is a nuclear magnetic resonance hydrogen spectrum graph of compound 1 of the present application

[0022] Figure 5 is a nuclear magnetic resonance carbon spectrum graph of compound 1 of the present application DETAILED DESCRIPTION

[0023] Example 1:

[0024] (I) Preparation of compound 1

[0025] (1) Preparation of the strain

[0026] The purified culture medium used is composed of 200 parts of potato, 20 parts of glucose, 20 parts of agar and 1 L of purified water. After high-temperature sterilization, the slant is prepared and placed in a normal temperature state. When no miscellaneous bacteria grow, the mycelium of the fungus Sordariomycetes sp. GZWMJZ-1598 is inoculated and cultured at 28°C.

[0027] (2) Preparation of the inoculum

[0028] The volume parts of the inoculation medium used are 200 parts of potato, 20 parts of glucose and 1 L of purified water. After high-temperature sterilization, the liquid culture medium is prepared and placed in a normal temperature state. The strain obtained in step (1) is inoculated and cultured at 28°C at a speed of 180 r / min for 3 days as the inoculum.

[0029] (3) Inoculation

[0030] The solid fermentation method is adopted. 200 fermentation strain bags are prepared. Each strain bag is filled with 50 parts of rice and 50 parts of purified water. After high-temperature sterilization at 121°C for 30 min, the above-mentioned strain is inoculated and cultured at 28°C for 30 days.

[0031] (4) Extraction

[0032] The fermentation product and mycelium obtained by fermentation are soaked with ethyl acetate for 48 hours. Then, they are extracted in batches with a stirrer. Each batch is extracted three times, and each extraction lasts for 30 minutes. After standing, the supernatant is combined and recovered with an ethyl acetate rotary evaporator to obtain 350 grams of crude extract.

[0033] (4) Separation and purification of monomers

[0034] The dissolved crude extract was mixed with 100-200 mesh silica gel and subjected to silica gel column chromatography, gradient elution with petroleum ether-ethyl acetate (v / v = 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1) and gradient elution with dichloromethane-methanol (v / v = 10:1, 5:1), TLC detection, and the same fractions were combined to obtain 20 fractions. Fraction 15 (9.0 g) was subjected to HPLC analysis and gradient elution with methanol-water containing 1.0% trifluoroacetic acid (v:v = 20:70-100:0) using a Triptech rapid preparation system, TLC detection, to obtain fractions Fr.15-1 to Fr.15-10. Fr.15-4 (578.2 mg) was subjected to Toyopearl HW-40F gel column chromatography using methanol-dichloromethane (1:1, v / v) and the fractions were combined after TLC detection to obtain Fr.15-4-1 to Fr.15-4-6. Fr.15-4-1 (32.0 mg) was subjected to semi-preparative HPLC [mobile phase: acetonitrile-pure water solution (32:68, v / v), using a naphthalene ring column at a flow rate of 4 mL / min] to obtain compound 1 (10.9 mg).

[0035] Structure identification of compound 1

[0036] The structure of compound 1 was determined by comprehensive analysis of high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, optical rotation, nuclear magnetic resonance, single crystal X-ray diffraction, and the like, and the physicochemical properties thereof are as follows:

[0037] Compound 1: white powder; molecular formula C 13 H 22 O4; molecular weight 242; UV (MeOH) λ max (logε) 325 (4.27) nm; IR (KBr) v max 3412, 3119, 1400, 1048, 985 cm -1 ; HRESIMS m / z 265.1407 [M+Na] + (calculated for C 13 H 22 O7Na, 265.1410); (c 0.10, MeOH), and the nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum data thereof are shown in Table 1.

[0038] Table 1. Nuclear magnetic resonance hydrogen spectrum and carbon spectrum data of compound 1 (DMSO-d6)

[0039]

[0040] Example 2

[0041] To further verify the beneficial effects of the synthesized compounds of the present application, anti-inflammatory activity tests were performed on Compound 1, and the specific experiments are as follows:

[0042] Cell culture: The mouse monocyte macrophage cell line RAW264.7 was incubated and grown in a high-sugar DMEM medium containing 10% FBS at 37°C in a constant-temperature incubator with 5% CO2, and was subcultured every other day.

[0043] MTT method for detecting the effect of the extract on cell viability: The logarithmic growth phase RAW264.7 cells were diluted to 1×10 5 6 / mL with DMEM culture solution containing 10% fetal bovine serum, and inoculated in a 96-well culture plate at 200 μL per well, and cultured for 24 h. After the cells were completely adherent, different concentrations of samples were added to the drug administration groups, with a final concentration of 50, 25, 10, 5, and 1 μM. The solvent control groups did not add any test drugs and LPS, but added DMSO with a volume fraction of 0.1%. The zero adjustment group only added culture solution without cells for zero adjustment. Three replicate wells were set for each experimental concentration. After 24 h of incubation in the incubator, 15 μL of 5 mg / mL MTT solution was added to each well, and incubation was continued for 4 h. The supernatant was then aspirated, 150 μL of DMSO was added to each well, and transferred to a microplate reader for shaking for 10 min to fully dissolve the precipitates. The absorbance (A) value at 490 nm was detected by the microplate reader. The cell survival rate was calculated according to the following formula.

[0044] Cell survival rate = (A 给药 -A 调零 ) / (A 对照 -A 调零 )

[0045] Griess method for determining the effect of the compound on the amount of NO released by LPS-induced RAW2.2.3264.7 cells: The logarithmic growth phase RAW264.7 cells were diluted to 1×10 5Cells were seeded at a concentration of 1 μg / mL in 96-well plates, with a final volume of 200 μL per well. The plates were incubated at 37°C and 5% CO2 for 24 h. Experimental setups included a blank control group (no LPS or sample), an LPS group (final concentration 1 μg / mL), a positive control group (LPS + dexamethasone), a drug-treated group (LPS + sample), and a zeroing group. Each group had three replicates. After 24 h of incubation, the cultured cells were centrifuged, and the supernatant was mixed with an equal volume of Griess reagent (BEYOTIME Total Nitric Oxide Assay Kit) and incubated at room temperature for 10 min. The supernatant was then used to detect NO levels. The absorbance of each well was measured at 540 nm using a Thermo Scientific MULTISKAN MK3 microplate reader. Statistical analysis was performed using SPSS 20.0 software. The inhibition rate was calculated using the formula to obtain the half-maximal inhibitory concentration (IC50). 50 ).

[0046] Inhibition rate = (A LPS -A 给药 ) / (A LPS -A 空白 )

[0047] The results showed that compound 1 had no effect on the viability of RAW264.7 cells at concentrations of 50 μM and below; however, it inhibited the NO release of LPS-induced RAW264.7 inflammatory cells by an IC50 threshold. 50 The concentration was 0.83 ± 0.25 μM, significantly better than the positive control dexamethasone (IC50). 50 (5.04±0.28μM).

[0048] These embodiments should be understood as illustrative only and not as limiting the scope of protection of the invention. After reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent changes and modifications also fall within the scope defined by the claims of this invention.

Claims

1. A cyclohexenone compound, characterized in that, The compound has the structure shown in Formula 1. 。 2. A method for preparing the cyclohexenone compound as described in claim 1, characterized in that, Compound 1 was isolated and purified from the fermentation products of deep soil fungi, wherein the deep soil fungi were named: Sordariomycetes sp. GZWMJZ-1598 was deposited on June 6, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20251925. The preparation method of compound 1 includes the following steps: Preparation of strain A: The purified culture medium used consists of 200-300 parts by weight of potato, 20-25 parts by weight of glucose, and 15-20 parts by weight of agar. Add this to 1 L of purified water, adjust the pH to 6.5-7.5, prepare a slant, and inoculate at room temperature. Sordariomycetes The mycelium of sp. GZWMJZ-1598 was cultured at 25-32℃ for 3 days as a strain; B. Preparation of inoculum: Use 200-300 parts potato and 20-25 parts glucose as inoculum, add them to 1 L of purified water, adjust the pH to 6.5-7.5 to prepare a liquid culture medium, and inoculate the inoculum obtained in step A at room temperature. Incubate at 25-32 ℃ and 150-200 r / min for 3 days to obtain the inoculum. C. Inoculation: Using solid-state fermentation, each fermentation inoculum bag is filled with 30-100 parts rice and 30-100 parts purified water. The inoculum obtained in step B is inoculated and then incubated at 28 ℃ for 30 days to obtain the fermentation medium. D Extraction: Soak the fermentation medium described in step C in ethyl acetate for 48-72 hours, then stir and extract 3-5 times with a stirrer for 30-40 minutes each time. Combine the supernatants and recover ethyl acetate in a rotary evaporator to obtain crude extract. E. Monomer separation and purification: The crude extract obtained in step D is separated by column chromatography to obtain compound 1. The chromatographic separation method includes silica gel column chromatography, gel column chromatography, and semi-preparative high performance liquid chromatography.

3. The use of the cyclohexenone compound as described in claim 1 in the preparation of an anti-inflammatory drug.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the cyclohexenone compound of claim 1 and pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition contains 0.1–99% by mass of the cyclohexenone compound of claim 1, with the remainder being a pharmaceutical carrier or excipient.

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