Ionic mycin derivative as well as preparation method and application thereof

Abstract

The invention discloses an ionomycin derivative, the structure of which is selected from one of the following structures: # imgabs0 #. Two new structures generated by fermentation of a streptomycete S.conglobatus ATCC 31005 mutant strain RJ81 are found for the first time, namely, the ionomycin derivatives IOP-A and IOP-B with the new structures containing alpha-pyranoid ring groups. An anti-inflammatory experiment shows that the compounds IOP-A and IOP-B have obvious inhibitory activity on the expression level of IL-6 in LPS-stimulated THP-1 cells, and the IC50 values of the compounds IOP-A and IOP-B are 3.66 mu M and 4.47 mu M respectively. The anti-inflammatory activity of IOP-A is further verified in a mouse colitis model and no detectable injury is found in the heart, liver, lung and kidney of the IOP-A treated mouse.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and specifically relates to an ionomycin derivative, a preparation method thereof and an application thereof. Background Art

[0002] Carboxyl polyether natural molecules derived from Streptomyces can bind to cations and transport ions through hydrophobic membranes, representing a class of natural products with a wide range of biological activities, having antibacterial, anti-tumor and anti-parasitic properties (Expert Opin Drug Discov. 2009, 4(2):109–146). Their chemical structural characteristics are linear polyketones containing polycyclic ether bonds and terminal carboxyl groups, such as lasalocid, monensin, nigericin, salinomycin, nanchangmycin and ionomycin (Biomed Res Int. 2013:2013:162513) (as Figure 19 shown, Figure 19 is a schematic diagram of the structure of representative carboxyl polyether natural molecules derived from Streptomyces, and the enol structure of ionomycin is marked in red.). Compared with other carboxyl polyether structures, ionomycin contains a unique enol group ( Figure 19 ), and this group and the terminal carboxyl group participate in the affinity of ionomycin for calcium ions (Angew Chem Int Ed Engl. 2009, 48(27),5022-5025).

[0003] The molecular skeleton of ionomycin is biosynthesized by type I polyketide synthases (PKS) (as Figure 20 shown, Figure 20Among them, a) is the ionomycin biosynthetic gene cluster ino BGC (GenBank PQ613730), and b) is a schematic diagram of the deduced ionomycin biosynthetic pathway. Type I PKS is similar to fatty acid synthase, and each catalytic functional module (module, M) is responsible for the extension of a two-carbon unit of the polyketide chain (Biochemistry. 2014, 53(18):2875-83). One PKS functional module includes at least three catalytic domains: the β-ketoacyl synthase (KS) catalyzes the formation of the C-C bond, the acyltransferase (AT) loads the appropriate extension unit, and the acyl carrier protein (ACP) domain. The PKS module also contains non-essential catalytic functional domains, such as the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains, which increase the structural diversity by reducing the β-keto group of the polyketide extension chain (Beilstein J Org Chem. 2017, 13:348-371). In addition, the ionomycin biosynthetic pathway involves epoxidase and epoxide hydrolase responsible for the formation of the five-membered ether ring, and these ether ring-forming catalytic elements are conserved in the biosynthetic pathways of carboxyl polyether natural products (J. Am. Chem. Soc. 2012, 134, 7246−7249; Angew Chem Int Ed Engl. 2015, 54(46):13622-5; Nat Commun. 2023,14(1):6273).

[0004] Drugs for treating inflammatory bowel disease (IBD) mainly include salicylic acids, glucocorticoids, immunosuppressants, and monoclonal antibody-targeted drugs. Among them, in traditional clinical medications, 5-aminosalicylic acid is used for the induction of remission in mild active IBD, glucocorticoids are used for the induction of remission in moderate or severe active IBD, and immunosuppressants are for patients who are resistant or dependent on glucocorticoids. However, glucocorticoids have the side effect of severe adrenal gland damage, and about 20% - 30% of IBD patients are resistant to them (Gastroenterology, Vol. 16, No. 12, 2011). The immunosuppressants azathioprine and cyclosporine A have side effects such as myelosuppression and nephrotoxicity respectively (Clin Gastroenterol Hepatol, 2006, 4(6):760 - 765). The monoclonal antibody drug infliximab, which targets TNF-α and is developed in recent years for the treatment of IBD, can rapidly induce and maintain remission of CD symptoms, but it is found to have the side effect of causing pulmonary and upper respiratory tract infections (Int J Dig Dis, 2013, Vol. 33, No. 4), and as a biological macromolecule preparation, the medication cost is relatively expensive. Summary of the Invention

[0005] An object of the present invention is to provide an ionomycin derivative.

[0006] Another object of the present invention is to provide a preparation method of the ionomycin derivative.

[0007] Another object of the present invention is to provide an application of the ionomycin derivative in the preparation of drugs for treating inflammatory bowel disease.

[0008] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0009] In the first aspect of the present invention, an ionomycin derivative is provided, and the structure is selected from one of the following structures:

[0010]

[0011] In the second aspect of the present invention, a preparation method of the ionomycin derivative is provided, including the following steps:

[0012] Streptomyces RJ81 was inoculated on an SFM solid plate medium and cultured at 27 - 32 °C for 4 - 6 days, and then the spores were collected. The spores were inoculated into a first-stage liquid medium and cultured with shaking (220 rpm) at 27 - 32 °C (preferably 30 °C) for 2 - 4 days (preferably 3 days) to obtain a first-stage seed liquid; the first-stage seed liquid was inoculated into a second-stage liquid medium and cultured with shaking (220 rpm) at 27 - 32 °C (preferably 30 °C) for 2 - 4 days (preferably 3 days) to obtain a second-stage seed liquid; the second-stage seed liquid was inoculated into a fermentation medium and cultured with shaking (220 rpm) at 27 - 32 °C (preferably 30 °C) for 3 - 8 days (preferably 6 days) to obtain a fermentation broth;

[0013] Formic acid was added to the fermentation broth (the volume percentage of formic acid in the fermentation broth was 0.1%), and the fermentation broth was extracted with an equal volume of ethyl acetate at least three times, and then concentrated to obtain an extract; the extract was redissolved with methanol, filtered to remove residues, and the oil was removed with n-hexane; after the methanol solution was concentrated, it was passed through a normal-phase silica gel column (Sillica gel, 200 - 300 mesh) under reduced pressure to obtain 10 fractions A - J, and the target fraction was detected to be in fraction H by HPLC - MS;

[0014] The target fraction was separated by preparative MPLC and a reversed-phase ODS column (Santai Technologies, Inc., Spherical C18, 20 - 45 μm, 100 Å) to obtain the ionomycin derivative.

[0015] The SFM solid plate medium: 2% soybean powder, 2% D - mannitol, 2% agar.

[0016] The first-stage liquid medium: 3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract, 0.1% (v / v) antifoaming agent.

[0017] The second-stage liquid medium: 3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract, 0.1% (v / v) antifoaming agent.

[0018] The fermentation medium: 3% soybean powder, 5% glucose, 0.5% CaCO3, 0.2% (v / v) antifoaming agent.

[0019] The volume ratio of the first-stage seed liquid to the second-stage liquid medium was 1:20.

[0020] The volume ratio of the second-stage seed liquid to the fermentation medium was 1:20.

[0021] The elution solvent of the normal-phase silica gel column was dichloromethane - methanol, and the elution solvent ratio gradient was 50 / 1 to 0 / 1 (v / v).

[0022] The HPLC-MS conditions: Conventional HPLC-MS analysis was performed using Waters HPLC combined with an Acquity QDa detector. A Waters Xbridge C18 column (250 mm × 4.6 mm, 5 μm) was used, eluted with 75% acetonitrile (0.1% formic acid, v / v), with an elution time of 20 min and an elution volume of 0.8 mL / min. -1 。

[0023] The preparative MPLC and reverse ODS column separation conditions were: 15 mL / min, 10% - 100% acetonitrile (containing 0.1% formic acid).

[0024] Among the ionomycin derivatives, the one with a retention time of 150 min is IOP-A, and the one with a retention time of 170 min is IOP-B.

[0025] The method for preparing Streptomyces RJ81 includes the following steps:

[0026] Streptomyces S. conglobatus ATCC 31005 was genetically engineered to modify the ionomycin biosynthetic polyketide chain assembly line, and the ketoreductase KR11 of the 11th PKS module was site-directed mutagenized and inactivated to obtain the mutant strain RJ81.

[0027] Preferably, the method for preparing Streptomyces RJ81 includes the following steps:

[0028] First, the genes congE and natE related to the production of Conglobatin and Neoantimycin in S. conglobatus ATCC 31005 were knocked out by in-frame deletion to obtain the mutant strain RJ8 with a relatively clean fermentation background. On this basis, based on bioinformatics analysis, site-directed mutagenesis Y1370F was performed at Y1370 of the inoE gene related to ionomycin synthesis to inactivate the KR11 functional domain. According to the sequence alignment of KRs, this site acts as the Tyr residue in the YxxxN key active motif of the KR11 functional domain, and finally the mutant strain RJ81 was obtained.

[0029] In the third aspect of the present invention, there is provided an application of the ionomycin derivative in the preparation of an anti-inflammatory drug.

[0030] In the fourth aspect of the present invention, there is provided an application of the ionomycin derivative in the preparation of a drug for treating inflammatory bowel disease.

[0031] Anti-inflammatory experiments showed that compounds IOP-A and IOP-B had significant inhibitory activity on the expression level of IL-6 in LPS-stimulated THP-1 cells, and their IC 50 values were 3.66 and 4.47 μM, respectively. The anti-inflammatory activity of compound IOP-A was further verified in a mouse colitis model. In an acute mouse colitis model induced by 2.5% DSS, compound IOP-A showed excellent activity in alleviating weight loss, reducing colon shortening, and increasing survival rate. Compound IOP-A significantly alleviated inflammatory symptoms, such as reducing the level of IL-6 in mouse serum and restoring the increased spleen weight to normal levels. Histological analysis of colon tissues revealed the role of compound IOP-A in alleviating the structural damage of crypts, mucosa, and tissue edema induced by DSS. According to AB-PAS staining, compound IOP-A improved the secretion of acidic and neutral mucus, thus reducing the loss of the intestinal barrier after DSS induction. In immunohistochemical analysis, compound IOP-A increased the proportion of Ki67+ cells in the crypts damaged by DSS induction, thus restoring the regeneration and proliferation of epithelial cells. In addition, according to H&E staining analysis, no detectable damage was found in the hearts, livers, lungs, and kidneys of mice treated with compound IOP-A. It is indicated that compounds IOP-A and IOP-B can be used as drugs for the treatment of inflammatory bowel disease or as anti-inflammatory drugs.

[0032] Due to the adoption of the above technical solutions, the present invention has the following advantages and beneficial effects:

[0033] The present invention first discovered two new structures produced by fermentation of a mutant strain RJ81 of Streptomyces conglobatus ATCC 31005, namely the new structure ionomycin derivatives IOP-A and IOP-B containing α-pyran ring groups, and provided a preparation method. At present, most anti-IBD drugs have relatively large side effects, such as adrenal injury, bone marrow production inhibition, kidney toxicity, and side effects of lung and upper respiratory tract infections. The IC 50 values of compounds IOP-A and IOP-B against human THP-1 cells exceeded 100 μM ( Figure 16 shown in a), indicating no cytotoxicity. Anti-inflammatory experiments showed that compounds IOP-A and IOP-B had significant inhibitory activity on the expression level of IL-6 in LPS-stimulated THP-1 cells, and their IC 50 values were 3.66 and 4.47 μM ( Figure 16 shown in b). Treatment with compound IOP-A was non-toxic to different organs of DSS-induced colitis ( Figure 16as shown in Figure c). The pictures are representative H&E stainings of the heart, liver, lung, and kidney after treatment with solvent, 2.5% DSS, and 30 mg / kg IOP-A (n = 5) (scale bar: 50 μm). The anti-inflammatory activity of IOP-A was further verified in a murine colitis model. In an acute murine colitis model induced by 2.5% DSS, IOP-A showed excellent activity in alleviating weight loss ( Figure 17 as shown in Figure a), reducing colon shortening ( Figure 17 as shown in Figures b and c), and increasing survival rate ( Figure 17 as shown in Figure d). IOP-A significantly alleviated inflammatory symptoms, such as reducing the level of IL-6 in the serum of mice ( Figure 17 as shown in Figure e), and restoring the increased spleen weight to normal levels ( Figure 17 as shown in Figure f). Histological analysis of colon tissues revealed the role of IOP-A in alleviating the structural damage of crypts, mucosa, and tissue edema induced by DSS ( Figure 17 as shown in Figure g). According to AB-PAS staining, IOP-A improved the secretion of acidic and neutral mucus, thereby reducing the loss of the intestinal barrier after DSS induction. In immunohistochemical analysis, IOP-A increased the proportion of Ki67+ cells in the crypts damaged by DSS induction, thereby restoring the regeneration and proliferation of epithelial cells. In addition, according to H&E staining analysis, no detectable damage was found in the heart, liver, lung, and kidney of mice treated with IOP-A. It is indicated that IOP-A has the potential to become an anti-colitis or anti-inflammatory drug. Description of the Drawings

[0034] Figure 1 is a schematic diagram of 2D NMR (DMSO-d6 or CDCl3) correlations of compounds IOP-A and IOP-B.

[0035] Figure 2 is of compound IOP-A 1 1H-NMR schematic diagram.

[0036] Figure 3 is of compound IOP-A 13 13C-NMR schematic diagram.

[0037] Figure 4 is the DEPT 135 schematic diagram of compound IOP-A.

[0038] Figure 5 is of compound IOP-A 1 1H- 1 1H COSY schematic diagram.

[0039] Figure 6It is the HSQC schematic diagram of compound IOP-A.

[0040] Figure 7 It is the HMBC schematic diagram of compound IOP-A.

[0041] Figure 8 It is the NOESY schematic diagram of compound IOP-A.

[0042] Figure 9 It is of compound IOP-B 1 1H-NMR schematic diagram.

[0043] Figure 10 It is of compound IOP-B 13 13C-NMR schematic diagram.

[0044] Figure 11 It is the DEPT 135 schematic diagram of compound IOP-B.

[0045] Figure 12 It is of compound IOP-B 1 1H- 1 1H-1H COSY schematic diagram.

[0046] Figure 13 It is the HSQC schematic diagram of compound IOP-B.

[0047] Figure 14 It is the HMBC schematic diagram of compound IOP-B.

[0048] Figure 15 It is the NOESY schematic diagram of compound IOP-B.

[0049] Figure 16 It is the schematic diagram of the non-toxicity and anti-inflammatory activity test results of compounds IOP-A and IOP-B.

[0050] Figure 17 It is the schematic diagram of the anti-inflammatory activity test results of compound IOP-A in a mouse colitis model.

[0051] Figure 18 It is the production and activity pattern diagram of IOP-A, and the anti-inflammatory active molecule IOP-A is obtained from the KR-11 inactivated strain.

[0052] Figure 19 It is the schematic diagram of the results of representative Streptomyces-derived carboxyl polyether natural molecules.

[0053] Figure 20 Among them, a) is the ionomycin biosynthetic gene cluster ino BGC (GenBank PQ613730), and b) is the deduced schematic diagram of the ionomycin biosynthetic pathway.

[0054] Figure 21 It is a schematic diagram of the production pathways of IOP-A and IOP-B. The pyran ring is formed by premature termination of the assembly chain to obtain IOP-A and IOP-B. Detailed implementation mode

[0055] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific content described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0056] Depository description:

[0057] Strain name: RJ81;

[0058] Depository number: CCTCC NO. M 2025141;

[0059] Taxonomic nomenclature: Streptomyces conglobatus RJ81;

[0060] Depository date: January 15, 2025;

[0061] Depository institution: China Center for Type Culture Collection;

[0062] Address of depository institution: Wuhan University, Wuhan, China.

[0063] The Streptomyces S.conglobatus used in the following experiments is the commercially available strain ATCC 31005.

[0064] Example 1

[0065] The preparation method of ionomycin derivatives Ionpyrone A (IOP-A) and Ionpyrone B (IOP-B) includes the following steps:

[0066] The ionomycin biosynthetic polyketide chain assembly line of Streptomyces S. conglobatus ATCC 31005 was modified by genetic engineering, and the ketoreductase KR11 of the 11th PKS module was site-directed mutagenized and inactivated to obtain the mutant strain RJ81. Figure 18 It is a schematic diagram of the production and activity of IOP-A. The anti-inflammatory active molecule IOP-A was obtained from the KR-11 inactivated strain.

[0067] The specific method is as follows: First, the genes congE and natE related to the production of Conglobatin and Neoantimycin in S. conglobatus ATCC 31005 were knocked out by in-frame deletion to obtain the mutant strain RJ8 with a relatively clean fermentation background. On this basis, based on bioinformatics analysis, site-directed mutagenesis Y1370F was performed at Y1370 of the inoE gene related to Ionomycin synthesis to inactivate the KR11 domain. According to the sequence alignment of KRs, this site acts as the Tyr residue in the YxxxN key active motif of the KR11 domain, and finally the mutant strain RJ81 producing IOP-A and IOP-B was obtained (as Figure 21 shown, Figure 21 is the pathway pattern diagram of the production of IOP-A and IOP-B, where the assembly chain terminates prematurely to form a pyran ring to obtain IOP-A and IOP-B.).

[0069] (1) Fermentation of Streptomyces RJ81

[0070] Inoculate Streptomyces RJ81 on an SFM solid plate medium (2% soybean powder, 2% D-mannitol, 2% agar), culture at 27 - 32 °C for 4 - 6 days, collect spores with a cotton swab, dip a small amount of spores and inoculate them into a 50 mL spring-loaded Erlenmeyer flask containing 25 mL of the first-stage liquid medium (3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract, 0.1% (v / v) antifoaming agent), and culture with shaking at 30 °C and 220 rpm for 3 days to obtain the first-stage seed liquid. Inoculate the first-stage seed liquid at 1 / 20 (v / v) into a 500 mL spring-loaded Erlenmeyer flask containing 150 mL of the second-stage liquid medium (3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract, 0.1% (v / v) antifoaming agent), and culture with shaking at 30 °C and 220 rpm for 3 days to obtain the second-stage seed liquid. Inoculate the second-stage seed liquid at 1 / 20 (v / v) into a 500 mL spring-loaded Erlenmeyer flask containing 150 mL of the fermentation medium (3% soybean powder, 5% glucose, 0.5% CaCO3, 0.2% (v / v) antifoaming agent), and culture with shaking at 30 °C and 220 rpm for 6 days, collect and obtain 12 liters of fermentation broth.

[0071] (2) Extraction of IOP-A / IOP-B from Streptomyces fermentation products

[0072] Add 0.1% (v / v) formic acid to the fermentation broth, and extract the fermentation broth three times with an equal volume of ethyl acetate. The ethyl acetate extract is concentrated under reduced pressure at 40 °C to obtain an extract. The extract is redissolved in 300 mL of methanol, filtered through filter paper to remove residues, and the oil is removed twice with 200 mL of n-hexane through a separating funnel. After the methanol solution is concentrated, it is loaded onto a normal-phase silica gel column (Sillica gel, 200 - 300 mesh) by mixing the sample, and the column is passed through under reduced pressure. The elution solvent is dichloromethane - methanol, and the elution solvent ratio gradient is 50 / 1 to 0 / 1 (v / v). Ten fractions A - J are obtained. The fractions containing IOP-A / IOP-B are detected in fraction H by HPLC-MS (conventional HPLC-MS analysis is performed using Waters HPLC combined with an Acquity QDa detector. A Waters Xbridge C18 chromatographic column (250 mm × 4.6 mm, 5 μm) is used, eluted with 75% acetonitrile (0.1% formic acid, v / v), the elution time is 20 min, and the elution volume is 0.8 mL min-1).

[0073] The target fraction is finally separated using preparative MPLC and a reverse ODS column (Santai Technologies, Inc., SphericalC18, 20 - 45 μm, 100 Å). The separation conditions are: 15 mL / min, 10% - 100% acetonitrile (containing 0.1% formic acid), obtaining IOP-A with a retention time of 150 min, a light brown oil, 35 mg; and IOP-B with a retention time of 170 min, a light brown oil, 62 mg.

[0074] The structures of the compounds are shown as follows:

[0075]

[0076] Structure analysis of compounds IOP-A and IOP-B:

[0077] Take 1 - 10 mg of the sample and dissolve it in 0.5 mL of CDCl3 or DMSO-d6, and collect NMR data using an Aglient DD2 600 MHz NMR spectrometer. Through 1 1H-NMR and 13 13C-NMR data analysis to determine the compound structure, and use high-resolution mass spectrometry (HR-ESI-MS) analysis to determine the compound molecular formula.

[0078] Figure 1 It is a schematic diagram of the 2D NMR (DMSO-d6 or CDCl3) correlations of compounds IOP-A and IOP-B. Figures 2 - 15 They are respectively for compounds IOP-B and IOP-A1 1H-NMR, 13 13C-NMR, DEPT 135, 1 1H- 1 1H COSY, HSQC, HMBC, NOESY spectra. Figure 2 are the 1 1H-NMR schematic diagrams of compound IOP-A. Figure 3 are the 13 13C-NMR schematic diagrams of compound IOP-A. Figure 4 is the DEPT 135 schematic diagram of compound IOP-A. Figure 5 are the 1 1H- 1 1H COSY schematic diagrams. Figure 6 is the HSQC schematic diagram of compound IOP-A. Figure 7 is the HMBC schematic diagram of compound IOP-A. Figure 8 is the NOESY schematic diagram of compound IOP-A. Figure 9 are the 1 1H-NMR schematic diagrams of compound IOP-B. Figure 10 are the 13 13C-NMR schematic diagrams of compound IOP-B. Figure 11 is the DEPT135 schematic diagram of compound IOP-B. Figure 12 are the 1 1H- 1 1H COSY schematic diagrams. Figure 13 is the HSQC schematic diagram of compound IOP-B. Figure 14 is the HMBC schematic diagram of compound IOP-B. Figure 15 is the NOESY schematic diagram of compound IOP-B.

[0079] Tables 1 to 2 are the NMR analysis data of compounds IOP-A / IOP-B:

[0080] Table 1 NMR data of compound IOP-B

[0081] 1 1H-NMR (600 MHz, CDCl3) and 13 13C-NMR (151 MHz, CDCl3)

[0082]

[0083] Table 2 NMR data of compound IOP-A

[0084] 11H-NMR (600 MHz, DMSO-d6) and 13 13C-NMR (151 MHz, DMSO-d6)

[0085]

[0086]

[0087]

[0088] Example 2

[0089] Anti-inflammatory activity detection of ionomycin derivatives containing α-pyran ring groups, namely compounds IOP-A and IOP-B

[0090] Antibacterial activities of compounds IOP-A and IOP-B against Staphylococcus aureus, Bacillus mycoides, Mycobacterium smegmatis and Candida albicans. The minimum inhibitory concentration was determined by the 96-well plate method. The positive control compounds were vancomycin (for Staphylococcus aureus and Bacillus mycoides), rifampicin (for Mycobacterium smegmatis) and amphotericin B (for Candida albicans). DMSO was used as the negative control. Compounds IOP-A and IOP-B did not show antibacterial activities against the indicator strains Staphylococcus aureus, Bacillus mycoides, Mycobacterium smegmatis and Candida albicans.

[0091] Cell culture conditions: THP-1 cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and cultured in RPMI-1640 medium in an incubator containing 5% CO2. The medium was supplemented with 100 mg / mL streptomycin, 100 U / mL penicillin and 10% fetal bovine serum. To differentiate into macrophages, THP-1 cells were treated with 200 nM PMA (phorbol-12-myristate-13-acetate) for 48 hours.

[0092] Cell viability assay: THP-1 cells were seeded in 96-well plates at a density of 5×10³ cells / well for 24 hours. The cells were treated with different concentrations of the sample compounds for 24 hours. Then, 10 µl of CCK-8 reagent (Dojindo Laboratories) was added to each well. The mixture was incubated at 37 °C for 40 minutes, and then the absorbance was measured at 450 nm using a microplate reader.

[0093] ELISA for detecting cytokines: The differentiated THP-1 cells (5×10 5Cultured in 6-well plates, after stimulation with LPS (lipopolysaccharide) for 24 hours, the cells were treated with the sample compound for 2 hours. The expression level of IL-6 in the cell supernatant was analyzed using an IL-6 ELISA kit (BioLegend).

[0094] Establishment of a murine colitis model: Male C57BL / 6 mice (18 - 22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). To induce experimental colitis, C57BL / 6 mice were given 2.5% DSS (dextran sulfate sodium, MP Biomedicals) in drinking water for 7 consecutive days, followed by a 3-day recovery period with normal drinking water. All animal handling and procedures were performed in accordance with the institutional guidelines approved by the Animal Care Committee and the Animal Experiment Ethics Committee of Shanghai Jiao Tong University.

[0095] Treatment of compound IOP-A in a murine colitis model: A 10 mM DMSO solution of compound IOP-A was diluted with a solvent composed of 77.5% sterile saline, 20% PEG-400, and 2.5% TWEEN 80 to obtain a 3 mg / ml solution. The mice were randomly divided into five groups: control group, model group, model group + 5-ASA (5-aminosalicylic acid, 250 mg / kg), and model group + IOP-A (3 mg / kg and 30 mg / kg). In the DSS-induced colitis model, compound IOP-A was administered starting from day 2. Compound IOP-A or 5-ASA was injected intraperitoneally into the mice every two days. The body weight changes were recorded daily. On day 10, the mice were sacrificed by cervical dislocation to collect the colon and blood. The IL-6 level in the blood samples was analyzed using an ELISA kit. The colon tissues were examined by histological staining.

[0096] Histological analysis: The colon tissues were fixed with 10% formalin, then dehydrated and embedded in paraffin. The embedded tissue sections were stained with hematoxylin and eosin (HE). To examine the goblet cells in the epithelial layer, the paraffin-embedded sections were stained with PAS to identify neutral mucin.

[0097] Immunohistochemical staining: The colon samples were fixed with 4% paraformaldehyde. After dehydration and clearing, the tissues were embedded in paraffin. After antigen retrieval and peroxidase blocking, the slides were treated with 5% goat serum and incubated overnight with the Ki67 primary antibody. The sections were incubated with the secondary antibody and then subjected to DAB staining. The cell nuclei were counterstained with hematoxylin.

[0098] Statistical analysis: The results are expressed as mean ± SD. One-way ANOVA was used for statistical comparison between the treatment groups and the control group. A p-value ≤ 0.05 was considered to indicate a statistically significant difference.

[0099] The IC 50 values of compounds IOP-A and IOP-B against human THP-1 cells exceeded 100 μM ( Figure 16 as shown in a), indicating no cytotoxicity. Figure 16 is a schematic diagram of the non-toxic and anti-inflammatory activity detection results of compounds IOP-A and IOP-B. Among them, a) is a schematic diagram of the cytotoxicity results of compounds IOP-A and IOP-B against THP-1 cells.

[0100] Anti-inflammatory experiments showed that compounds IOP-A and IOP-B had significant inhibitory activity on the expression level of IL-6 in LPS-stimulated THP-1 cells, and their IC 50 values were 3.66 and 4.47 μM respectively ( Figure 16 as shown in b), and b) is a schematic diagram of the inhibitory effect of compounds IOP-A and IOP-B on the IL-6 expression of LPS-stimulated THP-1 cells;). (Ionomycin did not show anti-inflammatory activity and no further work was carried out)

[0101] Treatment with compound IOP-A was non-toxic to different organs of DSS-induced colitis ( Figure 16 as shown in c), and c) is a schematic diagram of the toxicity results of IOP-A on different organs of dss-induced colitis.). The pictures are representative H&E stains of the heart, liver, lung, and kidney after treatment with solvent, 2.5% DSS, and 30 mg / kg IOP-A (n = 5) (scale bar: 50 μm).

[0102] The anti-inflammatory activity of compound IOP-A was further verified in a mouse colitis model. Figure 17 is a schematic diagram of the anti-inflammatory activity test results of compound IOP-A in a mouse colitis model. Among them, a) is a schematic diagram of the body weight results; b-c) are schematic diagrams of the colon length results; d) is a schematic diagram of the survival rate results; e) is a schematic diagram of the serum IL-6 level results; f) is a schematic diagram of the spleen weight results; g) are the results of H&E, AB-PAS, and Ki67 in intestinal tissues. In an acute mouse colitis model induced by 2.5% DSS, compound IOP-A showed excellent activity in reducing weight loss ( Figure 17 as shown in a), reducing colon shortening ( Figure 17 as shown in b and c), and increasing the survival rate ( Figure 17 as shown in d).

[0103] Compound IOP-A significantly alleviated inflammatory symptoms, such as reducing the IL-6 level in the serum of mice ( Figure 17 as shown in e), and restoring the increased spleen weight to normal levels ( Figure 17 as shown in f).

[0104] Histological analysis of colon tissue revealed the role of compound IOP-A in alleviating the structural damage of crypts, mucosa, and tissue edema induced by DSS ( Figure 17 as shown in g). According to AB-PAS staining, compound IOP-A improved the secretion of acidic and neutral mucus, thereby reducing the loss of the intestinal barrier after DSS induction. In immunohistochemical analysis, compound IOP-A increased the proportion of Ki67+ cells in the crypts damaged by DSS, thus restoring the regeneration and proliferation of epithelial cells. In addition, according to H&E staining analysis, no detectable damage was found in the hearts, livers, lungs, and kidneys of mice treated with compound IOP-A.

[0105] The above are only the preferred embodiments of the present invention, and there is no limitation to the present invention in any form. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent can make some changes or modifications to equivalent embodiments with equivalent changes by using the technical content prompted above within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the present invention.

Claims

1. An ionomycin derivative, characterized in that The structure is selected from one of the following structures: 。 2. A method for preparing the ionomycin derivative according to claim 1, characterized in that: The following steps are involved: The Streptomyces RJ81 was inoculated on a SFM solid plate medium, and the spores were collected after culturing at 27-32°C for 4-6 days. The spores were inoculated into a primary liquid medium, and shake-cultured at 27-32°C for 2-4 days to obtain a primary seed liquid. Inoculate the primary seed solution into the secondary liquid culture medium, and culture at 27-32°C with shaking for 2-4 days to obtain the secondary seed solution; The secondary seed liquid is inoculated into the fermentation medium, and cultured with shaking at 27-32°C for 3-8 days to obtain the fermentation liquid; Formic acid is added to the fermentation broth, and the fermentation broth is extracted with an equal volume of ethyl acetate for at least three times, and concentrated to obtain an extract; the extract is redissolved with methanol, the residue is filtered out, and the oil is removed with n-hexane; after the methanol solution is concentrated, it is passed through a normal silica gel column under reduced pressure to obtain 10 fractions A-J, and the target fraction is detected by HPLC-MS to be present in the H fraction; The target fraction is separated by using preparative MPLC and reversed phase ODS column to obtain the ionomycin derivative.

3. The method for preparing an ionomycin derivative according to claim 2, characterized in that: The SFM solid plate medium: 2% soybean powder, 2% D-mannitol, 2% agar; The primary liquid culture medium includes: 3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract, and 0.1% defoaming agent.

4. The method for preparing an ionomycin derivative according to claim 2, characterized in that: The secondary liquid culture medium includes: 3% tryptic soy broth, 10.3% sucrose, 0.5% yeast extract, and 0.1% defoaming agent; The fermentation medium includes: 3% soybean powder, 5% glucose, 0.5% CaCO3, and 0.2% defoaming agent.

5. The method for preparing an ionomycin derivative according to claim 2, characterized in that: The volume ratio of the primary seed solution to the secondary liquid culture medium is 1:20; The volume ratio of the secondary seed liquid to the fermentation medium is 1:

20.

6. The method for preparing an ionomycin derivative according to claim 2, characterized in that: The elution solvent of the forward silica gel column is dichloromethane-methanol, and the elution solvent ratio gradient is 50 / 1 to 0 / 1.

7. The method for preparing an ionomycin derivative according to claim 2, characterized in that: The HPLC-MS conditions were as follows: conventional HPLC-MS analysis was performed using a Waters HPLC combined with an Acquity QDa detector, a Waters Xbridge C18 column, elution with 75% acetonitrile, elution time of 20 min, and elution volume of 0.8 mL min -1 ; The separation conditions of the preparative MPLC and reverse ODS column are: 15 mL / min, 10%-100% acetonitrile, containing 0.1% formic acid.

8. The method for preparing an ionomycin derivative according to claim 2, characterized in that: The preparation method of Streptomyces RJ81 comprises the following steps: The polyketide chain assembly line of ionomycin biosynthesis in Streptomyces S. conglobatus ATCC 31005 was modified through genetic engineering, and the ketoreductase KR11 of the 11th PKS module was inactivated by site-directed mutation to obtain the mutant strain RJ81.

9. Use of the ionomycin derivative according to claim 1 in the preparation of anti-inflammatory drugs.

10. Use of the ionomycin derivative according to claim 1 in the preparation of a drug for treating inflammatory bowel disease.

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