A meroterpenoid compound, its preparation method and application

Abstract

The present invention belongs to the technical field of microbial pharmacy, and specifically provides a diterpenoid compound, a preparation method and an application thereof. The structural formula of the diterpenoid compound is shown in formula (I). The preparation method is to inoculate the fungus Penicillium sp. GDGJ-285 into a PDA medium and a rice medium successively, and then extract the fermentation product with ethyl acetate. After concentration under reduced pressure, a crude extract is obtained; the crude extract is subjected to silica gel column chromatography, gradient elution with petroleum ether-ethyl acetate, reverse-phase C18 column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative HPLC, and finally separated. Experiments prove that the compound of the present invention has an IC50 value of 39.03 μM for inhibiting the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-induced mouse macrophage RAW 264.7 inflammation model, showing relatively significant anti-inflammatory activity and can be used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pharmaceuticals, and particularly relates to a diterpenoid compound, a preparation method thereof, and an application thereof. Background Art

[0002] Inflammation is a complex defensive response of organisms with a vascular system to injury factors. When a pro-inflammatory factor acts on the body, the body eliminates the pro-inflammatory factor through the inflammatory response, which is a process of injury and anti-injury. Many common diseases (such as autoimmune diseases, atherosclerosis, wound repair, etc.) belong to the category of inflammation. If inflammation is not controlled for a long time, gene mutations caused by immune cell-derived reactive oxygen species and various pathogenic mechanisms of the inflammatory response may lead to many diseases, including cancer, septic shock, diabetes, atherosclerosis, and arthritis, etc. Anti-inflammatory drugs are the second largest category of drugs clinically after anti-infective drugs. However, the clinical therapeutic effects of quite a number of anti-inflammatory drugs on the current market are not ideal enough, and some drugs have relatively large side effects. Therefore, discovering highly effective and low-toxic anti-inflammatory drugs has very important practical significance for human health. Endophytic fungi have the advantages of rich resources, unique metabolites, rapid growth, and renewability, and have become one of the important ways to discover novel anti-inflammatory compounds. Summary of the Invention

[0003] The object of the present invention is to provide a diterpenoid compound, a preparation method thereof, and an application thereof. The compound has significant anti-inflammatory activity and can be used to prepare anti-inflammatory drugs.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] One object of the present invention is to provide a diterpenoid compound, and the structural formula of the diterpenoid compound is shown as formula (I):

[0006]

[0007] The diterpenoid compound described in the present invention is isolated and prepared from the fermentation culture of the endophytic fungus Penicillium sp. GDGJ-285 of Sophora subprostrata. The endophytic fungus Penicillium sp. GDGJ-285 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC), located on the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou City, with the preservation number of GDMCC No: 61201 and the preservation date of September 21, 2020.

[0008] Another object of the present invention is to provide a preparation method of the above diterpenoid compound, including the following steps:

[0009] (1) Prepare the seed medium. Inoculate the endophytic fungus Penicillium sp. GDGJ-285 from Sophora subprostrata into the PDA medium and incubate it at 28 °C for 3 - 6 days to obtain the seed medium;

[0010] (2) Cut the seed medium obtained in step (1) into pieces the size of broad beans with an inoculation loop and inoculate it into the sterilized rice medium for fermentation culture for 30 - 60 days. Then soak the fermented product with ethyl acetate solvent for 1 - 5 times, combine the ethyl acetate extracts, and concentrate them under reduced pressure to obtain a crude extract;

[0011] (3) Subject the crude extract obtained in step (2) to silica gel column chromatography and perform gradient elution with petroleum ether - ethyl acetate. The gradient range of petroleum ether - ethyl acetate is: 100:0, 90:10, 70:30, 50:50, 30:70, 0:100. Collect the fraction Fr.3 eluted with a petroleum ether - ethyl acetate volume ratio of 70:30; Subject fraction Fr.3 to reverse-phase C18 column chromatography and elute it with a methanol - water volume ratio gradient of 30:70, 40:70, 50:50, 60:40, 70:30, 80:20, 100:0. Collect and combine the fraction eluted with a methanol - water volume ratio of 50:50 to obtain Fr.3.2; Subject Fr.3.2 to Sephadex LH-20 gel column chromatography and elute it with methanol with a volume fraction of 100% to obtain fraction Fr.3.2.2; Subject fraction Fr.3.2.2 to semi-preparative HPLC with a mobile phase of acetonitrile / water with a volume ratio of 52:48 and a flow rate of 2 mL / min. Collect the eluted part with a retention time of 23 min, and thus obtain the said diterpenoid compound, named peniclactone C.

[0012] Further, the components of the PDA medium in step (1) are: 200 g / L of potatoes, 20 g / L of glucose, and 15 - 20 g / L of agar powder.

[0013] Further, the endophytic fungus Penicillium sp. GDGJ-285 from Sophora subprostrata in step (1) is stored at -80 °C in a PDB - glycerol medium.

[0014] Further, the components of the rice medium in step (2) are: 50 - 90 g of rice and 120 - 150 mL of distilled water; The culture condition is static culture at room temperature.

[0015] Further, the soaking time for each soaking with ethyl acetate solvent in step (2) is 20 - 28 hours.

[0016] The third object of the present invention is to provide the application of the diterpenoid compound in the preparation of anti-inflammatory drugs.

[0017] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0018] 1. The present invention for the first time isolates and prepares a diterpenoid compound from the fermentation product of the fungus Penicillium sp. GDGJ-285. It is a novel compound with a six-ring (6 / 5 / 6 / 5 / 5 / 6) structure of ABCDEF. The IC 50 value of this compound, peniclactone C, for inhibiting the production of nitric oxide (NO) in the lipopolysaccharide (LPS)-induced inflammatory model of mouse macrophages RAW 264.7 is 39.03 μM, showing significant anti-inflammatory activity. It can be used for the preparation of anti-inflammatory drugs, providing candidate compounds for the research and development of new anti-inflammatory drugs and scientific basis for the development and utilization of natural active substances from terrestrial microorganisms.

[0019] 2. The preparation method of the present invention is simple and feasible, and is easy for large-scale industrial production. Detailed Embodiments

[0020] In order to more clearly illustrate the present invention, the following further illustrates the present invention through specific examples.

[0021] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further descriptions of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0022] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. It should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] The fungus Penicillium sp. GDGJ-285 in the embodiments of the present invention was isolated from Sophora subprostrata plants (east longitude 106°38′, north latitude 23°2′, altitude 648 m) collected from Jingxi City, Baise, Guangxi, China in October 2017. Through ITS sequence analysis and identification, the GenBank gene accession number is: MN636334. After blast comparison and homology analysis, this strain was identified as Penicillium sp. This strain has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC) located in Guangzhou, with the deposit number GDMCC No: 61201 and the deposit date of September 21, 2020.

[0024] I. Preparation of Compounds

[0025] Example 1

[0026] A method for preparing a meroterpenoid compound, comprising the following steps:

[0027] (1) Prepare a seed culture medium. The endophytic fungus Penicillium sp. GDGJ-285 of Sophora subprostrata used in this example was stored at -80 °C in a PDB-glycerol medium. When in use, first inoculate the endophytic fungus Penicillium sp. GDGJ-285 of Sophora subprostrata into a PDA medium and culture it at a constant temperature of 28 °C for 3 days to obtain a seed culture medium; the components of the PDB-glycerol medium are: 200 g / L of potatoes, 20 g / L of glucose, and 30% of glycerol; the components of the PDA medium are: 200 g / L of potatoes, 20 g / L of glucose, and 15 g / L of agar powder;

[0028] (2) Cut the seed culture medium obtained in step (1) into pieces the size of broad beans with an inoculation loop and inoculate it into a sterilized rice culture medium for fermentation culture for 30 days. The components of the rice culture medium are: 50 g of rice and 1200 mL of distilled water; a total of 90 conical flasks with a volume of 1000 mL are inoculated; the culture condition is static culture at room temperature;

[0029] Then, soak and extract the obtained solid fermented product once with an ethyl acetate solvent for 28 hours, and concentrate the ethyl acetate extract under reduced pressure to obtain a crude extract;

[0030] (3) Subject the crude extract obtained in step (2) to silica gel column chromatography with 300 meshes, and perform gradient elution with petroleum ether-ethyl acetate. The gradient range of the petroleum ether-ethyl acetate is: 100:0, 90:10, 70:30, 50:50, 30:70, 0:100. Detect each collected component by thin-layer chromatography, combine the same or similar parts, and obtain 6 components Fr.1-Fr.6. Collect the component Fr.3 eluted with a petroleum ether-ethyl acetate volume ratio of 70:30; subject the component Fr.3 to reverse-phase C18 column chromatography, and elute it with a methanol-water volume ratio of 30:70, 40:70, 50:50, 60:40, 70:30, 80:20, 100:0 in gradient. Detect each collected component by thin-layer chromatography, combine the same or similar parts, and obtain 4 sub-components Fr.3.1-Fr.3.4; collect and combine the component eluted with a methanol-water volume ratio of 50:50 to obtain Fr.3.2; subject Fr.3.2 to Sephadex LH-20 gel column chromatography and elute it with methanol with a volume fraction of 100% to obtain 3 sub-components Fr.3.2.1-Fr.3.2.3; subject the component Fr.3.2.2 to semi-preparative HPLC, with a mobile phase of acetonitrile / water with a volume ratio of 52:48 and a flow rate of 2 mL / min, and collect the eluted part with a retention time of 23 min, namely, the meroterpenoid compound is obtained, named peniclactone C.

[0031] Example 2

[0032] A method for preparing a meroterpenoid compound, comprising the following steps:

[0033] (1) Prepare a seed culture medium. The endophytic fungus Penicillium sp. GDGJ-285 of Sophora subprostrata used in this example was stored at -80 °C in a PDB-glycerol medium. When in use, first inoculate the endophytic fungus Penicillium sp. GDGJ-285 of Sophora subprostrata into a PDA medium and culture it at a constant temperature of 28 °C for 6 days to obtain a seed culture medium; the components of the PDB-glycerol medium are: 200 g / L of potatoes, 20 g / L of glucose, and 30% of glycerol; the components of the PDA medium are: 200 g / L of potatoes, 20 g / L of glucose, and 20 g / L of agar powder;

[0034] (2) Cut the seed culture medium obtained in step (1) into pieces the size of broad beans with an inoculation loop and inoculate it into a sterilized rice culture medium for fermentation culture for 60 days. The components of the rice culture medium are: 90 g of rice and 150 mL of distilled water; a total of 90 conical flasks with a volume of 1000 mL are inoculated; the culture condition is static culture at room temperature;

[0035] Then, soak and extract the obtained solid fermentation product 5 times with an ethyl acetate solvent, with each soaking time being 20 hours. After combining the ethyl acetate extracts and concentrating them under reduced pressure, a crude extract is obtained;

[0036] The crude extract obtained in step (2) was subjected to silica gel column chromatography with 400 mesh, and gradient elution was carried out with petroleum ether - ethyl acetate. The gradient range of petroleum ether - ethyl acetate was: 100:0, 90:10, 70:30, 50:50, 30:70, 0:100. Each collected fraction was detected by thin - layer chromatography, and the same or similar parts were combined to obtain 6 fractions, Fr.1 - Fr.6. The fraction Fr.3 eluted with a petroleum ether - ethyl acetate volume ratio of 70:30 was collected; the fraction Fr.3 was subjected to reverse - phase C18 column chromatography and eluted with a methanol - water volume ratio gradient of 30:70, 40:70, 50:50, 60:40, 70:30, 80:20, 100:0. Each collected fraction was detected by thin - layer chromatography, and the same or similar parts were combined to obtain 4 sub - fractions, Fr.3.1 - Fr.3.4; the fraction eluted with a methanol - water volume ratio of 50:50 was collected and combined to obtain Fr.3.2; Fr.3.2 was subjected to Sephadex LH - 20 gel column chromatography and eluted with 100% methanol by volume to obtain 3 sub - fractions, Fr.3.2.1 - Fr.3.2.3; the fraction Fr.3.2.2 was subjected to semi - preparative HPLC with a mobile phase of acetonitrile / water with a volume ratio of 52:48 and a flow rate of 2 mL / min. The eluted fraction with a retention time of 23 min was collected, and the heteroterpenoid compound was obtained, named peniclactone C.

[0037] II. Structure Identification of Compounds

[0038] The heteroterpenoid compound peniclactone C obtained in Examples 1 and 2 was tested for nuclear magnetic resonance, mass spectrometry, optical rotation, and X - ray single - crystal diffraction data to determine the structure of the compound. The results are as follows:

[0039] Compound peniclactone C: white crystal, melting point 273.9 - 275.9 °C, (concentration 0.1 μM / mL, solvent is methanol), 1 1H NMR (400 MHz) and 13 13C NMR (100 MHz) are shown in Table 1; high - resolution mass spectrometry [M + Na] + m / z is 477.1519 (the theoretical molecular formula and theoretical molecular weight are C 25 H 26 O8Na + , 477.1520).

[0040] Table 1 Compound peniclactone C 1 1H and 13 13C NMR data (400 MHz and 100 MHz, in DMSO)

[0041]

[0042]

[0043] The absolute configuration of the meroterpenoid peniclactone C was determined by combining X-ray single crystal diffraction. The single crystal data are shown in Table 2, and the crystal structure is shown in formula (I).

[0044] Table 2 Single crystal data of compound peniclactone C

[0045]

[0046]

[0047] III. Anti-inflammatory activity test

[0048] 1. The compounds prepared in Examples 1 and 2 of the present invention were subjected to an in vitro antifungal test, and macrophages RAW 264.7 induced by LPS were used as the research object for anti-inflammatory activity.

[0049] 2. Experimental method

[0050] (1) Cell culture

[0051] Add 3 ml of DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody to the culture flask. Dispense the cell suspension of macrophages RAW264.7 into each culture flask, gently blow and mix evenly, cover the lid of the culture flask, and place it in an incubator with a CO2 concentration of 5% and a temperature of 37 °C for culture. Observe the cell growth status every day. Observe the culture flask under an inverted microscope. When the cell growth density reaches about 85%, take out the cell culture flask and place it in a laminar flow hood. Discard the old medium, wash it 3 times with PBS, add 1 to 2 ml of DMEM high-glucose medium, scrape the adherent cells with a sterile cell scraper, gently blow and disperse the clumped cells to make them detached and mixed evenly. Discard a part of the cells to make the cell density about 5-10%, and add DMEM high-glucose medium for subculture. When we need to conduct subsequent experiments, take the cells in the logarithmic growth phase for the experiment.

[0052] (2) Determination of cell proliferation survival rate

[0053] After digesting the cells in the logarithmic growth phase, according to the density of 85% of each bottle of cells, they can be used to seed 4 96-well plates. Add a certain volume of high-glucose DMEM medium and gently blow to disperse evenly. Inoculate the cell suspension into the 96-well plates at a volume of 180 μL / well. When the cells adhere and the cell density reaches 70-80%, add 20 μL of the test drugs with different concentrations to each well. The test drugs are the compounds prepared in the examples of the present invention. After the test drugs act for 24 h, add 10 μL of 5 mg / mL MTT solution to each well. Incubate in a 37 °C constant temperature incubator for 4 h, then discard the supernatant. Add DMSO solution (150 μL / well) to dissolve formazan. Place the 96-well plates in an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at a wavelength of 490 nm; and calculate cell survival according to the following formula.

[0054]

[0055] Among them, A 样品 : Absorbance after adding the sample; A 空白 : Absorbance without adding the sample.

[0056] (3) Determination of NO content

[0057] After treating the cultured RAW 264.7 cells, inoculate them into 96-well plates at 180 μL / well and place them in an incubator for 12 h. After the culture is completed, discard the culture medium, add 180 μL / well of fresh medium and 20 μL / well of drugs with different concentrations (the test group uses the compounds prepared in the examples of the present invention, the positive group uses dexamethasone, and the blank control group does not add drugs but adds 20 μL / well of DMEM). Set 5 parallel replicates for each concentration, place them in a constant temperature incubator, add 10 μL / well of LPS after 1 h, and continue to culture for 24 h. Collect the cell supernatant, centrifuge, and take 50 μL each and add them to a new 96-well plate. Determine the content of NO according to the steps of the nitric oxide kit, measure the absorbance at a wavelength of 540 nm, and calculate the inhibition rate according to the following formula to calculate the IC 50 value. The successful establishment of the inflammation model follows that the OD value A LPS组 / A 空 is between 3 and 8, indicating successful stimulation. Use dexamethasone as the positive drug.

[0058] NO inhibition rate (%) = [(A LPS组 -A 空白组 ) / (A LPS组 -A 空白组 )]×100%

[0059] 3. Experimental results:

[0060] The IC value of the meroterpenoid peniclactone C prepared by the present invention for inhibiting the production of nitric oxide (NO) in the lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophage inflammation model is 39.03 ± 0.14 μM. The IC value of the positive control dexamethasone for inhibiting the production of nitric oxide (NO) in the LPS-induced RAW 264.7 murine macrophage inflammation model is 16.61 ± 1.41 μM (see Table 2). These results indicate that the meroterpenoid peniclactone C of the present invention has significant anti-inflammatory activity. The present invention provides candidate compounds for the research and development of new anti-inflammatory drugs and scientific basis for the development and utilization of natural active substances derived from terrestrial microorganisms. 50 The IC value of the positive control dexamethasone for inhibiting the production of nitric oxide (NO) in the LPS-induced RAW 264.7 murine macrophage inflammation model is 16.61 ± 1.41 μM (see Table 2). These results indicate that the meroterpenoid peniclactone C of the present invention has significant anti-inflammatory activity. The present invention provides candidate compounds for the research and development of new anti-inflammatory drugs and scientific basis for the development and utilization of natural active substances derived from terrestrial microorganisms. 50 value is 16.61 ± 1.41 μM (see Table 2). These results indicate that the meroterpenoid peniclactone C of the present invention has significant anti-inflammatory activity. The present invention provides candidate compounds for the research and development of new anti-inflammatory drugs and scientific basis for the development and utilization of natural active substances derived from terrestrial microorganisms.

[0061] Table 2 Inhibitory effect of compound peniclactone C on NO in LPS-induced RAW 264.7 cells

[0062]

[0063] The above description is a detailed description of the preferred and feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. A meroterpenoid compound, characterized in that: The structural formula of the diterpenoid compound is shown in Formula (I):

2. The preparation method of a meroterpenoid compound according to claim 1, wherein, The diterpenoid compound is isolated and prepared from the fermentation culture of the endophytic fungus Penicillium sp. GDGJ-285 in Sophora subprostrata. The preservation number of the endophytic fungus Penicillium sp. GDGJ-285 is GDMCC No: 61201. Its preparation method includes the following steps: (1) Prepare a seed medium. Inoculate the endophytic fungus Penicillium sp. GDGJ-285 in Sophora subprostrata into a PDA medium and incubate it at a constant temperature of 28 °C for 3 to 6 days to obtain a seed medium; (2) Cut the seed medium obtained in step (1) into pieces the size of broad beans with an inoculation loop and inoculate it into a sterilized rice medium for fermentation culture for 30 to 60 days. Then soak the fermentation product with ethyl acetate solvent for 1 to 5 times, combine the ethyl acetate extracts, and concentrate them under reduced pressure to obtain a crude extract; (3) The crude extract obtained in step (2) is subjected to silica gel column chromatography and gradient elution is carried out with petroleum ether-ethyl acetate. The gradient range of petroleum ether-ethyl acetate is: 100:0, 90:10, 70:30, 50:50, 30:70, 0:

100. Collect the fraction Fr.3 eluted with a petroleum ether-ethyl acetate volume ratio of 70:30; Subject the fraction Fr.3 to reverse-phase C18 column chromatography and gradient elute it with a methanol-water volume ratio of 30:70, 40:70, 50:50, 60:40, 70:30, 80:20, 100:

0. Collect and combine the fraction eluted with a methanol-water volume ratio of 50:50 to obtain Fr.3.2; Fr.3.2 is subjected to Sephadex LH-20 gel column chromatography and eluted with methanol with a volume fraction of 100% to obtain the fraction Fr.3.2.2; The fraction Fr.3.2.2 is subjected to semi-preparative HPLC, the mobile phase is acetonitrile / water with a volume ratio of 52:48, the flow rate is 2 mL / min, and the elution part with a retention time of 23 min is collected to obtain the diterpenoid compound.

3. The preparation method of a meroterpenoid compound according to claim 2, characterized in that: The components of the PDA medium described in step (1) are: 200 g / L of potatoes, 20 g / L of glucose, and 15 - 20 g / L of agar powder.

4. The preparation method of a meroterpenoid compound according to claim 2, wherein: The endophytic fungus Penicillium sp. GDGJ-285 in Sophora subprostrata described in step (1) is stored at -80 °C in a PDB-glycerol medium.

5. The preparation method of a meroterpenoid compound according to claim 2, characterized in that: The components of the rice medium described in step (2) are: 50 - 90 g of rice and 120 - 150 mL of distilled water; The culture condition is static culture at room temperature.

6. The preparation method of a meroterpenoid compound according to claim 2, characterized in that: The soaking time for each soaking with ethyl acetate solvent described in step (2) is 20 - 28 hours.

7. Use of the diterpenoid compound according to claim 1 in the preparation of an anti-inflammatory drug.

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