CN112812125A - Novel skeleton heteroterpene compound and preparation method and application thereof - Google Patents

Novel skeleton heteroterpene compound and preparation method and application thereof Download PDF

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CN112812125A
CN112812125A CN202011102302.1A CN202011102302A CN112812125A CN 112812125 A CN112812125 A CN 112812125A CN 202011102302 A CN202011102302 A CN 202011102302A CN 112812125 A CN112812125 A CN 112812125A
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杨瑞云
莫土香
黄锡山
张文秀
李俊
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Guangxi Normal University
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Abstract

The invention belongs to the technical field of microbial pharmacy, and particularly provides a novel skeleton heteroterpene compound and a preparation method and application thereof. The preparation method comprises the steps of sequentially inoculating fungus Penicillium sp GDGJ-285 into a PDA culture medium and a rice culture medium, extracting a fermentation product with ethyl acetate, and concentrating under reduced pressure to obtain a crude extract; the crude extract is obtained by silica gel column chromatography, gradient elution with petroleum ether-ethyl acetate, reversed phase C18 column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative HPLC, and final separation. Experiments prove that the compound has an IC50 value of 39.03 mu M for inhibiting the generation of Nitric Oxide (NO) by a Lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 inflammation model, shows relatively obvious anti-inflammatory activity, and can be used for preparing anti-inflammatory drugs.

Description

Novel skeleton heteroterpene compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of microbial pharmacy, in particular to a novel skeleton heteroterpene compound and a preparation method and application thereof.
Background
Inflammation is a complex defense reaction of the living organism with the vascular system against damaging agents. When inflammatory factors act on the body, the body eliminates inflammatory factors through an inflammatory reaction, which is a process of injury and anti-injury. Many common diseases (e.g., autoimmune diseases, atherosclerosis, wound repair, etc.) belong to the category of inflammation. If inflammation is not controlled for a long time, the genetic mutation caused by the active oxygen of immune cells and various pathogenesis of inflammatory reaction can cause many diseases, including cancer, septic shock, diabetes, atherosclerosis and arthritis. Anti-inflammatory drugs are second only to anti-infective drugs clinically in the 2 nd main category. However, the clinical treatment effect of a considerable number of anti-inflammatory drugs on the market is not ideal, and part of the drugs have large side effects. Therefore, the discovery of the anti-inflammatory drug with high efficiency and low toxicity has very important practical significance to human health. Endophytic fungi have the advantages of rich resources, unique metabolites, rapid growth, renewability and the like, and become one of important ways for discovering novel anti-inflammatory compounds.
Disclosure of Invention
The invention aims to: aiming at the existing problems, the novel skeleton heteroterpene compound, the preparation method and the application thereof are provided, and the compound has obvious anti-inflammatory activity and can be used for preparing anti-inflammatory drugs.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
one of the objects of the present invention is to provide a novel skeletal diterpenoid compound, which has a structural formula shown in formula (I):
Figure BDA0002724046190000011
the hetero-terpene compound is separated and prepared from a fermentation culture of a subprostrate sophora endophytic fungus Penicillium sp.GDGJ-285, wherein the endophytic fungus Penicillium sp.GDGJ-285 is preserved in Guangdong province microbial culture collection center (GDMCC) of No. 59 building 5 of Michelia Tokyo No. 100, Guangzhou city, the preservation number is GDMCC No. 61201, and the preservation date is 2020, 9 and 21 days.
The invention also aims to provide a preparation method of the novel framework diterpenoid compound, which comprises the following steps:
(1) preparing a seed culture medium, taking the sophora subprostrata endophytic fungi Penicillium sp.GDGJ-285 to be accessed into a PDA culture medium, and culturing at the constant temperature of 28 ℃ for 3-6 days to obtain the seed culture medium;
(2) cutting the seed culture medium obtained in the step (1) into broad beans by using an inoculating ring, inoculating the broad beans into a sterilized rice culture medium, fermenting and culturing for 30-60 days, soaking the fermented product in an ethyl acetate solvent for 1-5 times, combining ethyl acetate extracting solutions, and concentrating under reduced pressure to obtain a crude extract;
(3) performing silica gel column chromatography on the crude extract obtained in the step (2), and performing gradient elution by using petroleum ether-ethyl acetate, wherein the gradient range of the petroleum ether-ethyl acetate is as follows: 100:0, 90:10, 70:30, 50:50, 30:70, 0:100, and collecting a component Fr.3 eluted at a petroleum ether-ethyl acetate volume ratio of 70: 30; subjecting fraction Fr.3 to reverse phase C18 column chromatography with methanol-water volume ratio of 30:70, 40: 70, 50:50, 60: 40, 70:30, 80: 20, 100: gradient elution is carried out on 0, and the volume ratio of collected and combined methanol to water is 50:50 to obtain Fr.3.2; fr.3.2 is subjected to Sephadex LH-20 gel column chromatography, and is eluted by methanol with the volume fraction of 100 percent to obtain a component Fr.3.2.2; and (3) performing semi-preparative HPLC on the component Fr.3.2.2, wherein a mobile phase is acetonitrile/water with a volume ratio of 52:48, the flow rate is 2mL/min, and an elution part with a retention time of 23min is collected to obtain the new-framework hetero-terpene compound named as penicallactone C.
Further, the PDA culture medium in the step (1) comprises the following components: 200g/L of potato, 20g/L of glucose and 15-20 g/L of agar powder.
Further, the sophora subprostrata endophytic fungus Penicillium sp GDGJ-285 in the step (1) is preserved in a PDB-glycerol culture medium at-80 ℃.
Further, the rice culture medium in the step (2) comprises the following components: 50-90 g of rice and 120-150 mL of distilled water; the culture conditions were static culture at room temperature.
Further, the soaking time of each soaking in the ethyl acetate solvent in the step (2) is 20-28 hours.
The invention also aims to provide the application of the novel skeleton heteroterpene compound in preparing anti-inflammatory drugs.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the novel framework heteroterpene compound is separated and prepared from a fermentation product of fungus Penicillium sp. GDGJ-285 for the first time, is a novel compound with an ABCDEF six-ring (6/5/6/5/5/6) structure, and the IC of the compound penictatone C for inhibiting the generation of Nitric Oxide (NO) in a Lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 inflammation model50The value is 39.03 mu M, has obvious anti-inflammatory activity, can be used for preparing anti-inflammatory drugs, provides candidate compounds for researching and developing new anti-inflammatory drugs, and provides scientific basis for developing and utilizing natural active substances from terrestrial microorganisms.
2. The preparation method is simple, convenient and feasible, and is easy for industrial mass production.
Detailed Description
In order that the invention may be more clearly expressed, the invention will now be further described by way of specific examples.
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The fungus Penicillium sp GDGJ-285 in the embodiment of the invention is separated from Sophora subprostrata (106 degrees 38 'east longitude, 23 degrees 2' northern latitude, and 648m) collected from Jingxi city, Guangxi, in 2017 and 10 months. Through ITS sequence analysis and identification, the GenBank gene accession number is MN636334, through blast comparison and homology analysis, the strain is identified as Penicillium sp. The strain is preserved in Guangdong province microorganism culture Collection (GDMCC) in Guangzhou city, with the preservation number of GDMCC No. 61201 and the preservation date of 2020, 9 and 21 days.
Preparation of Compounds
Example 1
A process for the preparation of a novel backbone heteroterpene compound comprising the steps of:
(1) preparing a seed culture medium, wherein the sophora subprostrata endophytic fungus Penicillium sp.GDGJ-285 used in the embodiment is stored in a PDB-glycerol culture medium at-80 ℃, when in use, firstly, the sophora subprostrata endophytic fungus Penicillium sp.GDGJ-285 is taken to be accessed into a PDA culture medium, and the PDA culture medium is obtained by constant temperature culture at 28 ℃ for 3 days; the PDB-glycerol medium comprises the following components: 200g/L of potato, 20g/L of glucose and 30% of glycerol; the PDA culture medium comprises the following components: 200g/L of potato, 20g/L of glucose and 15g/L of agar powder;
(2) cutting the seed culture medium obtained in the step (1) into broad bean size by using an inoculating ring, inoculating the broad bean size into a sterilized rice culture medium, and fermenting and culturing for 30 days, wherein the rice culture medium comprises the following components: 50g of rice and 1200mL of distilled water; conical flasks with the volume of 1000mL are inoculated with 90 flasks in total; the culture condition is static culture at room temperature;
soaking the obtained solid fermentation product in ethyl acetate solvent for 28 hr for 1 time, and concentrating the ethyl acetate extractive solution under reduced pressure to obtain crude extract;
(3) performing 300-mesh silica gel column chromatography on the crude extract obtained in the step (2), and performing gradient elution by using petroleum ether-ethyl acetate, wherein the gradient range of the petroleum ether-ethyl acetate is as follows: detecting each collected component by thin layer chromatography at 100:0, 90:10, 70:30, 50:50, 30:70, 0:100, combining the same or similar parts to obtain 6 components Fr.1-Fr.6, and collecting the component Fr.3 eluted at the petroleum ether-ethyl acetate volume ratio of 70: 30; subjecting fraction Fr.3 to reverse phase C18 column chromatography with methanol-water volume ratio of 30:70, 40: 70, 50:50, 60: 40, 70:30, 80: 20, 100: gradient eluting with 0, detecting each component by thin layer chromatography, and mixing identical or similar parts to obtain 4 subfractions Fr.3.1-Fr.3.4; collecting and combining methanol and water in a volume ratio of 50:50 to obtain Fr.3.2; fr.3.2 is subjected to Sephadex LH-20 gel column chromatography and is eluted by methanol with the volume fraction of 100 percent to obtain 3 subfractions Fr.3.2.1-Fr.3.2.3; and (3) performing semi-preparative HPLC on the component Fr.3.2.2, wherein the mobile phase is acetonitrile/water with the volume ratio of 52:48, the flow rate is 2mL/min, and the elution part with the retention time of 23min is collected to obtain the new-framework hetero-terpene compound named as penicilactone C.
Example 2
A process for the preparation of a novel backbone heteroterpene compound comprising the steps of:
(4) preparing a seed culture medium, wherein the sophora subprostrata endophytic fungus Penicillium sp.GDGJ-285 used in the embodiment is stored in a PDB-glycerol culture medium at-80 ℃, when in use, firstly, the sophora subprostrata endophytic fungus Penicillium sp.GDGJ-285 is taken to be accessed into a PDA culture medium, and the PDA culture medium is obtained by constant temperature culture at 28 ℃ for 6 days; the PDB-glycerol medium comprises the following components: 200g/L of potato, 20g/L of glucose and 30% of glycerol; the PDA culture medium comprises the following components: 200g/L of potato, 20g/L of glucose and 20g/L of agar powder;
(5) cutting the seed culture medium obtained in the step (1) into broad bean size by using an inoculating ring, inoculating the broad bean size into a sterilized rice culture medium, and fermenting and culturing for 60 days, wherein the rice culture medium comprises the following components: 90g of rice and 150mL of distilled water; conical flasks with the volume of 1000mL are inoculated with 90 flasks in total; the culture condition is static culture at room temperature;
soaking the obtained solid fermentation product in ethyl acetate solvent for 5 times, wherein the soaking time is 20 hours each time, and combining ethyl acetate extracting solutions and concentrating under reduced pressure to obtain a crude extract;
performing 400-mesh silica gel column chromatography on the crude extract obtained in the step (2), and performing gradient elution by using petroleum ether-ethyl acetate, wherein the gradient range of the petroleum ether-ethyl acetate is as follows: detecting each collected component by thin layer chromatography at 100:0, 90:10, 70:30, 50:50, 30:70, 0:100, combining the same or similar parts to obtain 6 components Fr.1-Fr.6, and collecting the component Fr.3 eluted at the petroleum ether-ethyl acetate volume ratio of 70: 30; subjecting fraction Fr.3 to reverse phase C18 column chromatography with methanol-water volume ratio of 30:70, 40: 70, 50:50, 60: 40, 70:30, 80: 20, 100: gradient eluting with 0, detecting each component by thin layer chromatography, and mixing identical or similar parts to obtain 4 subfractions Fr.3.1-Fr.3.4; collecting and combining methanol and water in a volume ratio of 50:50 to obtain Fr.3.2; fr.3.2 is subjected to Sephadex LH-20 gel column chromatography and is eluted by methanol with the volume fraction of 100 percent to obtain 3 subfractions Fr.3.2.1-Fr.3.2.3; and (3) performing semi-preparative HPLC on the component Fr.3.2.2, wherein the mobile phase is acetonitrile/water with the volume ratio of 52:48, the flow rate is 2mL/min, and the elution part with the retention time of 23min is collected to obtain the new-framework hetero-terpene compound named as penicilactone C.
Structural identification of compounds
The new skeleton hetero terpene compound penictalane C obtained in the examples 1 and 2 is subjected to nuclear magnetism, mass spectrum, optical rotation and X-ray single crystal diffraction data tests to determine the structure of the compound, and the results are as follows:
compound penicillactone C: white crystals with a melting point of 273.9-275.9 ℃,
Figure BDA0002724046190000051
(the concentration is 0.1 mu M/mL solvent is methanol),1h NMR (400MHz) and13c NMR (100MHz) is shown in Table 1; high resolution mass spectrum [ M + Na]+m/z is 477.1519 (theoretical molecular formula and theoretical molecular weight are respectively C25H26O8Na+,477.1520)。
TABLE 1 Compound penicilactone C1H and13c NMR data (400MHz and 100MHz in DMSO)
Figure BDA0002724046190000052
Figure BDA0002724046190000061
The absolute configuration of the novel framework diterpenoid compound peniclactone C is determined by combining X-ray single crystal diffraction, the single crystal data of the novel framework diterpenoid compound peniclactone C are shown in a table 2, and the crystal structure is shown in a formula (I).
TABLE 2 Single Crystal data of the Compound penicilactone C
Figure BDA0002724046190000062
Figure BDA0002724046190000071
Figure BDA0002724046190000072
Anti-inflammatory Activity assay
1. The compounds prepared in examples 1 and 2 were subjected to an in vitro antifungal assay using LPS-induced macrophage RAW264.7 as the subject of anti-inflammatory activity.
2. Experimental methods
(1) Cell culture
Adding 10% fetal calf serum and 1% double antibody DMEM high sugar medium 3ml into culture bottle, subpackaging macrophage RAW264.7 cell suspension into each culture bottle, gently blowing off and mixing, covering the cover of the culture bottle, and placing in CO2The cells were cultured in an incubator at 37 ℃ at a concentration of 5% and the growth of the cells was observed every day. Observing the culture bottle under an inverted microscope, taking out the culture bottle when the cell growth density reaches about 85%, placing the culture bottle in an ultraclean workbench, discarding the old culture medium, washing with PBS for 3 times, adding 1-2 ml of DMEM high-sugar culture medium, scraping the adherent cells with an aseptic cell scraper, slightly blowing the scraped cells into clumps, and enabling the cells to fall off and be uniformly mixed. Discarding a part of cells to make the cell density about 5-10%, and adding DMEM high-sugar medium to allow passage. When we need to do later period experiment, we take the cells in logarithmic growth phase to do experiment.
(2) Cell proliferation viability assay
After the cells in the logarithmic growth phase are digested, the cells with the density of 85 percent in each bottle can be used for seeding 4 pieces of 96-well plates, a certain volume of DMEM high-sugar medium is added, and the cells are blown off gently and uniformly. The cell suspension was seeded in 96-well plates at a volume of 180. mu.L/well. When the cells adhere to the wall and the cell density reaches 70-80%, 20 mu L of drugs to be detected with different concentrations are added into each hole, and the drugs to be detected are the compounds prepared by the embodiment of the invention. After the drug to be detected acts for 24 hours, adding 10 mu L of 5mg/mLMTT solution into each hole, incubating in a constant-temperature incubator at 37 ℃ for 4 hours, removing the supernatant, adding DMSO solution (150 mu L/hole) to dissolve Jia Zan, and placing a 96-hole plate in an enzyme-labeling instrument to measure the OD value at the 490nm wavelength; and cell survival was calculated according to the following formula.
Figure BDA0002724046190000081
Wherein A isSample (I): absorbance after adding the sample; a. theBlank space: absorbance of no added sample.
(3) Determination of the NO content
Cultured RAW264.7 cells were treated and inoculated into a 96-well plate, added at 180. mu.L/well per well, and cultured in an incubator for 12 hours. After the culture is finished, the culture solution is discarded, a new culture medium 180 mu L/hole and medicines with different concentrations 20 mu L/hole are added (the test group adopts the compound prepared by the embodiment of the invention, the positive group adopts dexamethasone, the blank control group does not add 20 mu L/hole DMEM), 5 parallel multiple holes are arranged at each concentration, the test group is placed in a constant temperature incubator, 10 mu L/hole LPS is added after 1h, the culture is continued for 24h, cell supernatant is collected, 50 mu L of the cell supernatant is respectively added into a new 96-hole plate after centrifugation, the NO content is measured according to the steps of a nitric oxide kit, the absorbance is measured at the wavelength of 540nm, the inhibition rate is calculated according to the following formula, and IC is calculated50The value is obtained. Successful establishment of the inflammation model complies with OD value ALPS group/AAir conditionerBetween 3 and 8, for successful stimulation. Dexamethasone is used as a positive drug.
NO inhibition (%) ═[(ALPS group-ABlank group)/(ALPS group-ABlank group)]100％
3. The experimental results are as follows:
IC (integrated Circuit) for inhibiting generation of Nitric Oxide (NO) by using novel framework heteroterpene compound peniclactone C prepared by the invention to Lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 inflammation model50The value was 39.03. + -. 0.14. mu.M. IC of positive control dexamethasone for inhibiting Nitric Oxide (NO) generation of LPS (lipopolysaccharide) induced mouse macrophage RAW264.7 inflammation model50The value was 16.61. + -. 1.41. mu.M (see Table 2). This result shows that: the novel skeleton hetero-terpene compound penicilactone C has obvious anti-inflammatory activity. The invention provides a candidate compound for researching and developing new anti-inflammatory drugs and provides scientific basis for developing and utilizing natural active substances from terrestrial microorganisms.
TABLE 2 inhibitory Effect of the Compound penicilactone C on NO in LPS-induced RAW264.7 cells
Figure BDA0002724046190000082
Figure BDA0002724046190000091
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims (9)

1. A novel skeletal diterpenoid compound characterized in that: the structural formula of the heteroterpene compound is shown as a formula (I):
Figure FDA0002724046180000011
2. a novel skeletal heteroterpene compound according to claim 1, wherein: the heteroterpene compound is separated and prepared from a fermentation culture of a subprostrate sophora endophytic fungus Penicillium sp.GDGJ-285, and the accession number of the endophytic fungus Penicillium sp.GDGJ-285 is GDMCC No: 61201.
3. The process for the preparation of a novel skeletal diterpene compound according to claim 1, comprising the steps of:
(1) preparing a seed culture medium, taking the sophora subprostrata endophytic fungi Penicillium sp.GDGJ-285 to be accessed into a PDA culture medium, and culturing at the constant temperature of 28 ℃ for 3-6 days to obtain the seed culture medium;
(2) cutting the seed culture medium obtained in the step (1) into broad beans by using an inoculating ring, inoculating the broad beans into a sterilized rice culture medium, fermenting and culturing for 30-60 days, soaking the fermented product in an ethyl acetate solvent for 1-5 times, combining ethyl acetate extracting solutions, and concentrating under reduced pressure to obtain a crude extract;
(3) performing silica gel column chromatography on the crude extract obtained in the step (2), and performing gradient elution by using petroleum ether-ethyl acetate, wherein the gradient range of the petroleum ether-ethyl acetate is as follows: 100:0, 90:10, 70:30, 50:50, 30:70, 0:100, and collecting a component Fr.3 eluted at a petroleum ether-ethyl acetate volume ratio of 70: 30; subjecting fraction Fr.3 to reverse phase C18 column chromatography with methanol-water volume ratio of 30:70, 40: 70, 50:50, 60: 40, 70:30, 80: 20, 100: gradient elution is carried out on 0, and the volume ratio of collected and combined methanol to water is 50:50 to obtain Fr.3.2; fr.3.2 is subjected to Sephadex LH-20 gel column chromatography, and is eluted by methanol with the volume fraction of 100 percent to obtain a component Fr.3.2.2; and (3) performing semi-preparative HPLC on the component Fr.3.2.2, wherein a mobile phase is acetonitrile/water with a volume ratio of 52:48, the flow rate is 2mL/min, and an elution part with a retention time of 23min is collected to obtain the new-framework diterpenoid compound.
4. The process for the preparation of a novel skeletal diterpene compound according to claim 3, which comprises: the PDA culture medium in the step (1) comprises the following components: 200g/L of potato, 20g/L of glucose and 15-20 g/L of agar powder.
5. The process for the preparation of a novel skeletal diterpene compound according to claim 3, which comprises: the sophora subprostrata endophytic fungus Penicillium sp. GDGJ-285 in the step (1) is stored in a PDB-glycerol culture medium at the temperature of minus 80 ℃.
6. The process for the preparation of a novel skeletal diterpene compound according to claim 3, which comprises: the rice culture medium in the step (2) comprises the following components: 50-90 g of rice and 120-150 mL of distilled water; the culture conditions were static culture at room temperature.
7. The process for the preparation of a novel skeletal diterpene compound according to claim 3, which comprises: and (3) soaking the mixture in the ethyl acetate solvent for 20-28 hours each time.
8. Use of a novel skeletal diterpene compound according to claim 1 for the preparation of an anti-inflammatory medicament.
9. Use of the novel skeletal diterpene compounds prepared by the process according to claim 3 for the preparation of anti-inflammatory agents.
CN202011102302.1A 2020-10-14 2020-10-14 Novel skeleton heteroterpene compound and preparation method and application thereof Pending CN112812125A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115181083A (en) * 2022-08-11 2022-10-14 中国科学院华南植物园 Preparation method of compound Cyophiliolines A-B and application of compound Cyophiliolines A-B in preparation of anti-inflammatory drugs

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115181083A (en) * 2022-08-11 2022-10-14 中国科学院华南植物园 Preparation method of compound Cyophiliolines A-B and application of compound Cyophiliolines A-B in preparation of anti-inflammatory drugs
CN115181083B (en) * 2022-08-11 2023-10-10 中国科学院华南植物园 Preparation method of compound Cyophiobiolins A-B and application of compound Cyophiobiolins A-B in preparation of anti-inflammatory drugs

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