Compound containing 7-hydroxychromone structure and preparation method and application thereof

Abstract

The present invention belongs to the technical field of small molecule compounds, and discloses a compound with a 7-hydroxychromone structure, a preparation method and an application thereof. The compound has a structure shown in the following formula (I). The preparation method of the compound comprises the following steps: accessing the marine fungus sclerotium Gibberella to a seed culture medium, culturing on a shaking table, and obtaining a seed culture solution; accessing the seed culture solution to a fermentation medium, culturing statically, and obtaining a fermentation product; extracting the fermentation product statically three times with ethyl acetate, extracting the obtained bacteria three times with methanol, concentrating the extract to obtain an extract, and then using column chromatography separation technology to obtain a compound with a structural formula as shown in formula (I). The compound has the activity of inhibiting the production of nitric oxide in LSP-induced macrophages RAW264.7, and its IC50 is 38.26 μM, respectively, and can be used to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of small molecule compounds, and particularly relates to a compound with a 7-hydroxychromone structure and a preparation method and application thereof. Background Art

[0002] Due to its unique living environment: high salt, high pressure, low temperature and lack of oxygen, marine organisms have formed unique metabolic pathways and physical and chemical defense mechanisms, making them an important source of marine drug small molecules. For example, ziconotide, the first truly marine-derived drug approved by the US FDA in 2004 (derived from the South Pacific marine snail Conus magus), has been approved to date. More than ten marine-derived drugs have been approved, and a variety of marine-derived natural products are in Phase I, II, and III clinical trials. This shows that the ocean is an important source of new drugs in the future, and marine natural products have great development prospects.

[0003] Literature research found that many scholars at home and abroad have been studying the biological functions of chromones and their glycosides. The structure shows that they have anti-tumor, anti-inflammatory, analgesic, blood pressure lowering and other biological activities that are beneficial to human health. For example, the chromone glycoside component of Saposhnikovia divaricata has a significant effect on reducing the inflammatory response to skin swelling caused by xylene.

[0004] Therefore, it is of great significance to screen out compounds containing chromone structure. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a compound with a 7-hydroxychromone structure.

[0006] Another object of the present invention is to provide a method for preparing the compound having the 7-hydroxychromone structure.

[0007] Another object of the present invention is to provide an application of the compound having the 7-hydroxychromone structure.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A compound with a 7-hydroxychromone structure, wherein the structural formula (I) of the compound is as follows:

[0010]

[0011] The compound is isolated from the fermentation liquid of the marine fungus Gibberella sclerotiae, which is the marine fungus Gibberella sclerotiae with the preservation number of GDMCC No:60215.

[0012] The above-mentioned method for preparing a compound having a 7-hydroxychromone structure comprises the following steps:

[0013] S1. Inoculate the marine fungus Gibberella sclerotiorum into the seed culture medium and culture it in a shaking table to obtain a seed culture solution;

[0014] S2. inoculating the seed culture solution into the fermentation medium, culturing statically, and obtaining a fermentation product;

[0015] S3. The fermentation product is statically extracted with ethyl acetate three times, the obtained bacteria are extracted with methanol three times, the extract is concentrated to obtain an extract, and then column chromatography separation technology is used to obtain a compound with a structural formula as shown in formula (I).

[0016] The marine fungus Gibberella sclerotiorum described in step S1 is the marine fungus Gibberella sclerotiorum with a preservation number of GDMCC No: 60215; the formula of the seed culture medium is: on average, each liter of the seed culture medium includes 100 grams of glucose, 10 grams of yeast extract, 0.5 grams of magnesium sulfate, 1 gram of potassium dihydrogen phosphate, 5 grams of sodium chloride, and the balance is water.

[0017] The shaking culture conditions in step S1 are shaking speed of 200 rpm and culture at 25-30° C. for 3-7 days.

[0018] The formula of the fermentation medium in step S2 is: on average, each liter of the fermentation medium includes 20 grams of glucose, 2 grams of yeast extract, 0.1 grams of magnesium sulfate, 0.2 grams of potassium dihydrogen phosphate, 5 grams of sodium chloride, and the balance is water.

[0019] The static culture conditions in step S2 are 25-30° C. for 25-50 days.

[0020] The specific operation of the column chromatography separation in step S3 is: the extract is subjected to column chromatography separation using a silica gel column, and gradient elution is performed using dichloromethane-methanol with a volume ratio of 100:1 to 70:30 as an eluent, and the dichloromethane-methanol eluate with a volume ratio of 80:20 is collected, and then repeated column chromatography separation is performed using a normal phase silica gel column; the obtained eluate is then chromatographically separated using a semi-preparative liquid chromatography column chromatography, and a methanol aqueous solution with a volume percentage concentration of 60% is prepared and collected to obtain a compound with a 7-hydroxychromone structure.

[0021] Application of the above-mentioned compound with 7-hydroxychromone structure in the preparation of anti-inflammatory drugs.

[0022] Compared with the prior art, the present invention has the following advantages and effects:

[0023] Natural products have more significant structural diversity and complexity than synthetic compounds, and have a higher screening rate in terms of biological activity; marine microbial secondary metabolites show high novelty and significant biological activity, such as antibacterial, anti-tumor, immunomodulatory, anti-inflammatory, enzyme inhibition, etc., and have become an important resource for the research and development of marine medicinal natural products. The present invention provides a marine fungus-derived compound, which has the activity of inhibiting the production of nitric oxide (NO) in LSP-induced macrophages RAW264.7, and its IC50 is 38.26μM, respectively, and can be used to prepare anti-inflammatory drugs. Therefore, the marine fungus-derived compound provided by the present invention has clinical application potential for the treatment of inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the high resolution mass spectrum (HR-ESI-EI) of the compound obtained in Example 1.

[0025] Figure 2 The compound obtained in Example 1 1 H NMR.

[0026] Figure 3 The compound obtained in Example 1 13 C NMR. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific examples. However, the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment of the present invention are conventional reagents, methods and equipment in the art.

[0028] Example 1: Preparation of compounds

[0029] 1. Isolating and preparing a compound from the metabolites of the marine fungus Gibberella sclerotiorum with a deposit number of GDMCC No: 60215, the steps are as follows:

[0030] (1) Seed culture of the marine fungus Gibberella sclerotiorum with a deposit number of GDMCC No: 60215:

[0031] Seed culture medium composition: 20 grams of glucose, 2 grams of yeast extract, 0.1 grams of magnesium sulfate, 0.2 grams of potassium dihydrogen phosphate, 5 grams of sodium chloride, add water to 1L (specific preparation method: mix and dissolve each component and shake well).

[0032] After the culture medium is sterilized at high temperature, the strain is picked up with an inoculation loop and inoculated, and cultured in a constant temperature shaker at 25°C for 3 days to obtain the seed culture solution.

[0033] (2) Fermentation culture of marine fungus Gibberella sclerotiorum:

[0034] The fermentation medium formula is 20 grams of glucose, 2 grams of yeast extract, 0.1 grams of magnesium sulfate, 0.2 grams of potassium dihydrogen phosphate, and 5 grams of sodium chloride. Add deionized water to 1L, stir until completely dissolved, and divide into 500ml Erlenmeyer flasks. Stir and sterilize at 121°C for 20 minutes.

[0035] Transfer the strain in the seed solution into a conical flask filled with culture medium, with an inoculation volume of 8-20 ml, and always control the temperature at 28°C for 30 days.

[0036] (3) The fermentation product was subjected to static extraction with ethyl acetate three times, the obtained bacteria were extracted with methanol three times, and the extract was concentrated to obtain an extract; the extract was separated by column chromatography, and during separation, a gradient elution of dichloromethane-methanol with a volume ratio of 100:1 to 70:30 was used, and the dichloromethane-methanol eluate with a volume ratio of 80:20 was collected, and then repeated column chromatography separation was performed on a normal phase silica gel column; the obtained eluate was then chromatographically separated by semi-preparative liquid chromatography column chromatography, and prepared and collected with a methanol aqueous solution with a volume percentage concentration of 60%, to obtain the target compound.

[0037] Example 2: Structural test analysis of compounds

[0038] The compound obtained in Example 1 was subjected to structural test and analysis to obtain the following test data:

[0039] Compound: white powder, HR-ESI-MS m / z 263.1274[M+H] + (Theoretical value 263.1283, C 15 H 18 O4). One-dimensional NMR data: 1 H NMR 0.98(t,14.7,7.3),1.28(d,6.2),1.59(m),2.65(dd,14.3,8.0),2.72(dd,14.3,4.9) ,3.13(dd,9.1,6.4),4.19,(ddd,8.0,6.2,4.8),6.06(s),6.64(d,2.4),6.67(d,2.4); 13 C NMR 14.4, 23.5, 25.8, 38.3, 44.2, 66.4, 101.9, 112.5, 115.2, 117.7, 148.3, 161.8, 163.2, 166.9, 181.5.

[0040] The high resolution mass spectrum (HR-ESI-EI) of the compound obtained in Example 1 is as follows Figure 1 .

[0041] Example 1 The compound obtained 1 H NMR Figure 2 .

[0042] Example 1 The compound obtained 13 C NMR Figure 3 .

[0043] After identification, the structural formula of the compound is shown in the following formula (I):

[0044]

[0045] Example 4: Compound Activity Test

[0046] Experiment on the inhibitory effect of the compound obtained in Example 1 on NO production in RAW264.7 cells:

[0047] 1. Materials:

[0048] 1.1DMEM high-glucose medium, fetal bovine serum, trypsin (Gibco, USA); phosphate buffered saline (PBS) (Gibco, USA); dimethyl sulfoxide (MPbio, USA); CCK8 cell counting kit (APExBIO, USA); dexamethasone, lipopolysaccharide (LPS) (Sigma, USA); Griess kit (Beyond Biotech, Shanghai, China).

[0049] 1.2 Compound cytotoxicity test:

[0050] The compound was dissolved in dimethyl sulfoxide (100%) to a concentration of 20 mmol / L -1 Store in refrigerator for later use. Dilute to 200 μmol·L with DMEM high-glucose culture medium during experiment. -1 RAW264.7 cells were digested with 0.25% trypsin and resuspended in DMEM high-glucose medium containing 10% FBS at a concentration of 1.25×10 4 Cells (100 μL per well) were inoculated into 96-well plates and cultured in a 37°C constant temperature incubator containing 5% CO2 for 24 h. The culture medium of each group was replaced with fresh DMEM high-glucose culture medium containing 10% FBS. The compound test group was added with the corresponding compound (the compound obtained in Example 1 or dexamethasone) (10 μL per well), and the normal control group was added with DMEM high-glucose culture medium containing 10% fetal bovine serum (10 μL per well) and continued to culture for 24 h. Then, 5 mg·mL -1 MTT (10 μL per well) was used for live cell staining, and resveratrol was used as a control. After incubation for 3 hours, the culture medium was discarded, DMSO (100 μL per well) was added to dissolve, and it was shaken on a shaker to fully dissolve, and then the OD value was measured at a wavelength of 490 nm. 100 μL DMSO was added to each well of the blank group, and the cell survival rate of each group was calculated using the following formula:

[0051] Cell survival rate (%) = (OD value 化合物测试组 -OD value 空白组 ) / (OD value 正常对照组 -OD value 空白组 )×100%.

[0052] 1.3 Anti-inflammatory activity test of compounds

[0053] RAW264.7 cells were cultured at 2 × 10 per ml 5 The cells were inoculated at a density of 100 μL per well on a 96-well culture plate and cultured in a 37°C constant temperature incubator containing 5% CO2 for 24 hours. The culture medium of each group was replaced with fresh DMEM high-glucose culture medium containing 10% fetal bovine serum. The corresponding concentration of the test compound (the compound obtained in Example 1 or dexamethasone) was added to the drug administration group (10 μL per well), and the normal control group and the lipopolysaccharide model group were added with DMEM high-glucose culture medium containing 10% fetal bovine serum (10 μL per well). Subsequently, 0.001 mg·mL -1 The normal control group was added with 10 μL of lipopolysaccharide (10 μL per well), and the normal control group was added with 10 μL of DMEM high-glucose culture medium containing 10% FBS. After 24 hours of continuous culture, 50 μL of supernatant and 50 μL of Greiss reagent were taken from each well and reacted at room temperature for 15 minutes. The OD value was measured at a wavelength of 540 nm, and the molar mass fraction of nitric oxide (NO) in each group was calculated using the standard curve of nitric oxide, and the inhibition rate of each drug-added group was further calculated. The calculation formula is as follows:

[0054] NO generation inhibition rate (%) = 1-w1÷w2×100%

[0055] w1 represents the NO molar mass fraction of the compound test group, and w2 represents the NO molar mass fraction of the lipopolysaccharide group.

[0056] 2. Test results

[0057] The test results showed that the two cyclic peptide compounds (compounds obtained in Example 1 or dexamethasone) can significantly and effectively inhibit the production of NO by cells. The inhibitory effect of the two cyclic peptide compounds on the production of NO by RAW264.7 cells was IC 50 The values ​​(μM) are shown in Table 1.

[0058] Table 1 Inhibitory effects of two cyclic peptide compounds on NO production in RAW264.7 cells (IC 50 μM)

[0059]

[0060] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a compound having a 7-hydroxychromone structure, characterized in that The steps include: S1. The marine fungus Gibberella sclerotiorum is connected to the seed culture medium and cultured on a shaking table to obtain a seed culture solution; the marine fungus Gibberella sclerotiorum is a marine fungus Gibberella sclerotiorum with a deposit number of GDMCC No: 60215; The formula of the seed culture medium is as follows: on average, per liter of the seed culture medium includes 100 grams of glucose, 10 grams of yeast extract, 0.5 grams of magnesium sulfate, 1 gram of potassium dihydrogen phosphate, 5 grams of sodium chloride, and the balance is water; The shaking culture conditions are as follows: the shaking speed is 200 rpm, and the temperature is 25-30° C. for 3-7 days; S2. inoculating the seed culture solution into the fermentation medium, culturing statically, and obtaining a fermentation product; The formula of the fermentation medium is as follows: on average, per liter of the fermentation medium includes 20 grams of glucose, 2 grams of yeast extract, 0.1 g of magnesium sulfate, 0.2 grams of potassium dihydrogen phosphate, 5 grams of sodium chloride, and the balance is water; The static culture condition is 25-30° C. for 25-50 days; S3. The fermentation product is subjected to static extraction with ethyl acetate three times, the obtained bacterial cells are extracted with methanol three times, the extract is concentrated to obtain an extract, and then column chromatography separation technology is used to obtain a compound with a structural formula as shown in formula (I); the specific operation of the column chromatography separation is: the extract is subjected to column chromatography separation with a silica gel column, and gradient elution is performed with dichloromethane-methanol in a volume ratio of 100:1 to 70:30 as an eluent, and the dichloromethane-methanol eluate in a volume ratio of 80:20 is collected, and then repeated column chromatography separation is performed with a normal phase silica gel column; the obtained eluate is then subjected to chromatographic separation by semi-preparative liquid chromatography column chromatography, and is prepared and collected with a methanol aqueous solution with a volume percentage concentration of 60%, to obtain a compound with a 7-hydroxychromone structure; （Ⅰ）。

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