Dienopyrrolidinocidin and preparation method and application thereof

Abstract

The application belongs to the technical fields of compound preparation and biochemical medicine, and particularly relates to a dienopyrrol oxalicin, a preparation method and application thereof. The dienopyrrol oxalicin provided by the application is a new alkaloid compound, has strong anti-inflammatory activity, is important, has no cytotoxicity compared with conventional alkaloid compounds, is easy to synthesize, can be used for preparing medicines for preventing or treating inflammation, provides a new compound or small molecule lead compound for developing prevention or treatment of inflammation, and also provides a new drug source molecule with application value for the medical industry.

Description

Technical Field

[0001] This invention belongs to the field of compound preparation and biochemical pharmaceutical technology, specifically relating to a dienepyrroloxamic acid bacteriocin, its preparation method and application. Background Technology

[0002] Inflammation is a major threat to human health. In recent decades, researchers both domestically and internationally have been dedicated to developing highly effective and low-toxicity anti-inflammatory drugs. Penicillium is a type of ascomycete whose mycelium is typically multinucleate, septate, and consists of a highly branched network of colorless hyphae. Almost all Penicillium species possess potential medicinal value, dating back to Alexander Fleming's accidental discovery of penicillin in 1928. Microorganisms, due to their lack of seasonal growth restrictions and the ability to be cultivated on a large scale artificially, do not cause ecological damage compared to other biological resources, and their ease of source material acquisition makes them uniquely advantageous for subsequent research and development.

[0003] Alkaloids are a class of nitrogen-containing basic organic compounds found in nature. They are synthesized from different amino acids or their direct derivatives and are secondary metabolites. They are also an important active ingredient in traditional Chinese medicine. According to literature reports, alkaloids have various biological activities such as anti-inflammatory, anti-tumor, and antibacterial effects. However, most alkaloids, while exerting their pharmacological activities, also have toxic or strong physiological effects on organisms. Summary of the Invention

[0004] The purpose of this invention is to provide a dienepyrroloxamic acid compound, its preparation method and application. The dienepyrroloxamic acid compound is a new alkaloid compound with good anti-inflammatory activity and no cytotoxicity.

[0005] This invention provides a dienepyrroloxamic acid bactericide with the structure shown in Formula I:

[0006]

[0007] The present invention also provides a method for preparing the dienepyrroloxamic acid bacteriocin described in the above technical solution, comprising: subjecting a polar organic solvent extract of the fermentation product of the endophytic fungus Penicillium oxalicum MWTG-03 to silica gel column chromatography, gel column chromatography and high performance liquid chromatography in sequence to obtain the dienepyrroloxamic acid bacteriocin;

[0008] The eluent for the silica gel column chromatography is a dichloromethane-methanol system; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is 100:0 to 3:1.

[0009] The eluent for the gel column chromatography separation is methanol;

[0010] The mobile phase for separation by high performance liquid chromatography is a methanol-water system;

[0011] The preservation number of the Penicillium endophytic fungus MWTG-03 is CCTCC M 20231220.

[0012] Preferably, the silica gel column chromatography includes:

[0013] Elution was performed sequentially using dichloromethane-methanol systems with volume ratios of 100:0, 100:1, 50:1, 30:1, 10:1, and 3:1. The elution fraction with a volume ratio of 10:1 dichloromethane-methanol solution was collected to obtain the silica gel column chromatography solution.

[0014] Preferably, the volume ratio of methanol to water in the methanol-water system is 6-9:4-5.

[0015] Preferably, the high-performance liquid chromatography separation includes preparative high-performance liquid chromatography separation; the chromatographic column used in the high-performance liquid chromatography separation is a C18 column.

[0016] Preferably, the preparation of the polar organic solvent extract includes the following steps:

[0017] The activated Penicillium endophytic fungus MWTG-03 was inoculated into a fermentation medium for fermentation culture to obtain the fermentation product of the Penicillium endophytic fungus MWTG-03.

[0018] The fermentation product of the Penicillium endophytic fungus MWTG-03 was mixed with a polar organic solvent and then subjected to ultrasonic extraction to obtain a polar organic solvent extract.

[0019] Preferably, the fermentation medium is sterilized potato; the fermentation temperature is 18-28℃ and the time is 28-32 days.

[0020] Preferably, the mass ratio of the Penicillium endophytic fungus MWTG-03 fermentation product to the volume ratio of the polar organic solvent is (30-80) g: (50-150) mL; the ultrasonic extraction frequency is 40-53 kHz, and the time is 30-60 min.

[0021] The present invention also provides the application of dienepyrroloxamic acid bacteriocin as described in the above technical solution or the dienepyrroloxamic acid bacteriocin prepared by the preparation method described in the above technical solution in the preparation of anti-inflammatory drugs.

[0022] The present invention also provides a Penicillium oxalicum MWTG-03 endophytic fungus, the preservation number of which is CCTCC M 20231220.

[0023] Beneficial effects:

[0024] This invention provides a dienepyrroloxamic acid bactericide with the structure shown in Formula I:

[0025]

[0026]

[0027] The dienepyrroloxamic acid provided by this invention is a novel alkaloid compound with strong anti-inflammatory activity. Importantly, compared with conventional alkaloid compounds, it is not cytotoxic and can be used to prepare drugs for the prevention or treatment of inflammation. It provides new compounds or small molecule lead compounds for the development of drugs for the prevention or treatment of inflammation, and also provides new drug source molecules with application value for the medical industry.

[0028] The present invention also provides a method for preparing the dienepyrroloxamic acid bacteriocin, comprising: sequentially subjecting an ethyl acetate extract of the fermentation product of the endophytic fungus MWTG-03 (Penicillium oxalicum) to silica gel column chromatography, gel column chromatography, and high performance liquid chromatography to obtain the dienepyrroloxamic acid bacteriocin; the eluent for the silica gel column chromatography is a dichloromethane-methanol system; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is 100:0 to 3:1; the eluent for the gel column chromatography is methanol; the eluent for the high performance liquid chromatography is a methanol-water system; the preservation number of the endophytic fungus MWTG-03 is CCTCC M 20231220. The dienepyrroloxamic acid bacteriocin described in this invention is a metabolite of the endophytic fungus MWTG-03 of the Penicillium genus. It can be prepared by microbial fermentation. The preparation method has advantages such as mild culture conditions, simple and easy culture medium preparation, short fermentation time, and ease of large-scale production. It not only meets the needs of modern environmental protection and low-carbon economy, but also solves the raw material problem by being used to develop drugs for the prevention or treatment of inflammation.

[0029] Biological Preservation Information:

[0030] The endophytic fungus MWTG-03 (Penicillium oxalicum) was deposited on July 6, 2023, at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC M 20231220. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0032] Figure 1Molecular systematics analysis of the endophytic fungi isolated in Example 1 based on the maximum likelihood method of rpb2 and ITS sequences;

[0033] Figure 2 The colony characteristics of the endophytic fungi isolated in Example 1;

[0034] Figure 3 The key HMBC for dienepyrroloxamic acid bacteriocin in Example 3, 1 H- 1 H COSY correlation and single-crystal diffraction results;

[0035] Figure 4 The HR-ESI-MS spectrum of dienepyrroloxamic acid in Example 3;

[0036] Figure 5 The anti-inflammatory compound dienepyrroloxamic acid bacteriocin in Example 3 1 H-NMR spectrum;

[0037] Figure 6 The anti-inflammatory compound dienepyrroloxamic acid bacteriocin in Example 3 13 C-NMR and DEPT spectra;

[0038] Figure 7 The anti-inflammatory compound dienepyrroloxamic acid bacteriocin in Example 3 1 H- 1 H COSY spectrum;

[0039] Figure 8 The HSQC spectrum of dienepyrroloxamic acid bacteriocin, the anti-inflammatory active compound in Example 3;

[0040] Figure 9 The HMBC spectrum of dienepyrroloxamic acid bacteriocin, the anti-inflammatory active compound in Example 3;

[0041] Figure 10 The NOESY spectrum of dienepyrroloxamic acid bacteriocin, the anti-inflammatory active compound in Example 3. Detailed Implementation

[0042] This invention provides a dienepyrroloxamic acid bactericide with the structure shown in Formula I:

[0043]

[0044] The present invention also provides a method for preparing the dienepyrroloxamic acid bacteriocin described in the above technical solution, comprising: subjecting a polar organic solvent extract of the fermentation product of the endophytic fungus MWTG-03 (Penicillium oxalicum) to silica gel column chromatography, gel column chromatography and high performance liquid chromatography in sequence to obtain the dienepyrroloxamic acid bacteriocin;

[0045] The eluent for the silica gel column chromatography is a dichloromethane-methanol system; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is 100:0 to 3:1.

[0046] The eluent for the gel column chromatography separation is methanol;

[0047] The eluent used in the high-performance liquid chromatography separation is a methanol-water system;

[0048] The preservation number of the Penicillium endophytic fungus MWTG-03 is CCTCC M 20231220.

[0049] The present invention preferably prepares a ferment of Penicillium endophytic fungus MWTG-03. The preparation method of the ferment of Penicillium endophytic fungus MWTG-03 includes: inoculating activated Penicillium endophytic fungus MWTG-03 into a fermentation medium for fermentation culture to obtain the ferment of Penicillium endophytic fungus MWTG-03.

[0050] In this invention, the endophytic fungus MWTG-03 of the Penicillium genus is inoculated onto PDA slant medium for activation culture to obtain activated endophytic fungi of the Penicillium genus.

[0051] Figure 1 As shown, Figure 1 The name marked in red, MWTG-03, represents the endophytic fungus MWTG-03 of the genus Penicillium before it was named. Based on the similarity of 80% to 90%, it is considered to be at the same phylum level; 90% to 95% similarity is considered to be at the same family level; 95% to 97% similarity is considered to be at the same genus level; and above 97% similarity is considered to be at the same species level. This indicates that this strain has a very high similarity to Penicillium fungi. Therefore, this strain is named MWTG-03, an endophytic fungus of the genus Penicillium.

[0052] The endophytic fungus MWTG-03 of the Penicillium genus described in this invention exhibits a flat, velvety texture on the surface of its colonies in PDA medium; the conidia are densely packed, forming a deep conidial layer. When the petri dish is tapped, the conidial layer moves; the edge region of the colony is white in a 1-2 mm wide area, turning pale green, and becoming dark green as it moves towards the center of the colony; no exudate was observed, and there was no obvious odor.

[0053] The activation culture temperature of the present invention is preferably 18-28℃, more preferably 28℃; the time is preferably 3-5 days, more preferably 5 days.

[0054] After obtaining the activated Penicillium endophytic fungus MWTG-03, the present invention preferably inoculates the activated Penicillium endophytic fungus MWTG-03 onto a fermentation medium for fermentation culture to obtain the fermentation product of Penicillium endophytic fungus MWTG-03. The fermentation medium of the present invention is preferably sterilized potato. The preparation method of the fermentation medium of the present invention preferably includes: placing clean potato pieces in a tissue culture device for sealed sterilization to obtain the fermentation medium. The diameter of the clean potato pieces of the present invention is preferably 1 cm; the tissue culture device is preferably a tissue culture flask, and the mass of the clean potato pieces in the tissue culture flask is preferably 80-100 g / flask, more preferably 80 g / flask. The sterilization temperature of the present invention is preferably 120℃, and the time is preferably 30 min.

[0055] The fermentation culture temperature of the present invention is preferably 18-28℃, more preferably 28℃; the fermentation time is preferably 28-32 days, more preferably 30 days.

[0056] After obtaining the fermentation product of the Penicillium endophytic fungus MWTG-03, the present invention preferably involves mixing the fermentation product of the Penicillium endophytic fungus MWTG-03 with a polar organic solvent and then performing ultrasonic extraction to obtain an ultrasonic extract. The polar organic solvent of the present invention preferably includes ethyl acetate or methanol, more preferably ethyl acetate; the mass ratio of the fermentation product of the Penicillium endophytic fungus MWTG-03 to the volume ratio of ethyl acetate is preferably (30-80) g:(50-150) mL. The ultrasonic extraction power of the present invention is preferably 40-53 kHz, more preferably 53 kHz; the extraction time is preferably 30-60 min, more preferably 60 min.

[0057] After obtaining the ultrasonic extract, the present invention preferably performs solid-liquid separation on the ultrasonic extract, and concentrates the obtained extract under reduced pressure until solvent-free to obtain a polar organic solvent extract. The present invention does not specifically limit the specific process of solid-liquid separation and reduced pressure concentration; any process well known to those skilled in the art can be used, such as filtration for solid-liquid separation.

[0058] After obtaining the polar organic solvent extract, the present invention performs silica gel column chromatography on the polar organic solvent extract; the silica gel column chromatography preferably includes the following steps:

[0059] The polar organic solvent extract was dissolved in a dichloromethane-methanol solution to obtain a polar organic solvent extract solution.

[0060] The polar organic solvent extract solution is mixed with silica gel, the solvent in the resulting mixture is removed, and the resulting silica gel adsorbed with the polar organic solvent extract is packed into a column.

[0061] Gradient elution was performed sequentially using dichloromethane-methanol systems with volume ratios of 100:0, 100:1, 50:1, 30:1, 10:1, and 3:1. The 10:1 volume ratio segment was collected to obtain the silica gel column chromatography solution.

[0062] In this invention, the polar organic solvent extract is preferably dissolved in a dichloromethane-methanol solution to obtain a polar organic solvent extract solution. The volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution of this invention is preferably (1-5):1, more preferably 1:1.

[0063] After obtaining the polar organic solvent extract solution, the present invention preferably mixes the polar organic solvent extract solution with silica gel to remove the solvent from the resulting mixture, and then packs the resulting silica gel adsorbed with the polar organic solvent extract into a column. The preferred mass ratio of the polar organic solvent extract to silica gel in the present invention is (1-3):(1-2), more preferably 1.15:1. The preferred particle size of the silica gel in the present invention is 200 mesh. The present invention preferably uses vacuum concentration to remove the solvent from the resulting mixture; the vacuum concentration method is not particularly limited, and conventional vacuum concentration methods in the art are acceptable.

[0064] After the sample is packed into the column, the present invention preferably performs gradient elution with dichloromethane-methanol systems at volume ratios of 100:0, 100:1, 50:1, 30:1, 10:1, and 3:1, respectively, to obtain dichloromethane-methanol solution elution sections at volume ratios of 100:0, 100:1, 50:1, 30:1, 10:1, and 3:1; the dichloromethane-methanol solution elution section at a volume ratio of 10:1 is a silica gel column chromatography solvent. The preferred flow rate for the gradient elution in the present invention is 20–50 mL / min, more preferably 40 mL / min.

[0065] After obtaining the silica gel column chromatography solution, the present invention preferably performs gel column chromatography separation on the silica gel column chromatography solution to obtain a gel column chromatography eluent. The eluent used in the gel column chromatography separation of the present invention is preferably methanol; the flow rate is preferably 10-15 mL / min, more preferably 10 mL / min. The diameter of the pre-packed column used in the gel column chromatography separation of the present invention is preferably 2.5 cm, and the column length is preferably 2.0 m.

[0066] After obtaining the gel column chromatography eluent, the present invention preferably evaporates the gel column chromatography eluent to dryness using a rotary evaporator, dissolves it in methanol, filters it, and performs high-performance liquid chromatography (HPLC) separation to obtain the dienepyrroloxamic acid bacteriocin. The HPLC separation of the present invention is preferably preparative HPLC; the chromatographic column used for HPLC separation is preferably a C18 column, and the mobile phase preferably includes a methanol-water system, wherein the volume ratio of methanol to water in the methanol-water system is preferably 6-9:4-5, more preferably 6:4. The elution time of the present invention is preferably 7-10 min, and preferably the 8-min elution fraction is collected. The detection wavelength of the HPLC separation of the present invention is preferably 210 nm or 254 nm. The column temperature of the HPLC separation of the present invention is preferably 18℃; the flow rate of the mobile phase is preferably 2 mL / min. The dienepyrroloxamic acid bacteriocin prepared by the present invention is a novel alkaloid compound with strong anti-inflammatory activity. Importantly, compared with conventional alkaloid compounds, it does not exhibit cytotoxicity.

[0067] Based on the above-mentioned technical advantages, this invention also provides the application of the dienepyrroloxamic acid bacteriocin described in the above-mentioned technical solution or the dienepyrroloxamic acid bacteriocin prepared by the preparation method described in the above-mentioned technical solution in the preparation of anti-inflammatory drugs. The dienepyrroloxamic acid bacteriocin of this invention has a significant inhibitory effect on nitric oxide production in LPS-activated RAW264.7 macrophages, IC50... 50 The value was 28.14±1.58μM, indicating a good anti-inflammatory effect.

[0068] This invention also provides an endophytic fungus of the genus *Penicillium*, MWTG-03, whose preservation number is CCTCC M 20231220. The source and colony characteristics of the *Penicillium* endophytic fungus MWTG-03 described in this invention have been explained and defined in the above technical solutions and will not be repeated here.

[0069] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0070] Example 1

[0071] The steps for isolating and identifying the endophytic fungus *Penicillium oxalicum* are as follows:

[0072] (1) Preprocessing

[0073] Take freshly collected Aconitum carmichaelii roots (collected in Zhipu County, Guilin City, Guangxi Zhuang Autonomous Region; samples were immediately placed in sealed bags and stored at 4℃ after collection; isolation of endophytic fungi was completed within 72 hours after collection), remove surface mud and sand, and then rinse with tap water for about 2 hours;

[0074] (2) Surface disinfection

[0075] In a clean bench, the sample is sequentially soaked in a sodium hypochlorite solution with an effective chlorine content of 50%, then soaked in a 75% ethanol solution for 30 seconds, rinsed three times with sterile water, then soaked in a 75% ethanol solution for 30 seconds, rinsed four times with sterile water, and then dried with sterile filter paper before use.

[0076] To check whether the tissue surface disinfection is thorough, a control experiment is required. 50 μL of sterile water used for the last rinse is added directly to the isolation plate, and then spread on PDA medium with a spreader. The plate is then cultured under the same conditions as a control group. If no colonies grow in any of the control groups, it proves that the surface disinfection in the above steps is thorough, thus ensuring that the isolated microorganisms are plant endophytes rather than microorganisms adhering to the air or tissue surface.

[0077] (3) Isolation of endophytic fungi

[0078] The thoroughly disinfected root of Aconitum carmichaelii was cut into several 2×2mm pieces using a sterilized scalpel. 2 For small segments or pieces, implant the processed sample into pre-pouring PDA medium plates. Divide each plate into four regions (A, B, C, and D), and place 3-4 sample segments in each region. Seal the petri dishes with sealing film and label them. Invert them in a 28°C incubator and incubate daily. Once new colonies are observed, immediately inoculate them onto a new PDA medium plate.

[0079] (4) Purification of endophytic fungi (streaking method)

[0080] After mycelia grow on PDA culture medium plates, the grown mycelia are repeatedly streaked and cultured until a pure strain is obtained.

[0081] (5) Preservation of endophytic fungi

[0082] Short-term preservation of purified fungal strains can be achieved by directly inoculating them onto slant agar plates. After the strains have grown, the test tubes can be sealed with sealing film and stored in a refrigerator at 4°C.

[0083] (6) Identification of endophytic fungi

[0084] The endophytic fungus (tentative name MWTG-03) isolated in step (5) grew rapidly and had uniform edges when cultured on a PDA. At 28°C, it reached a diameter of 3.0 cm within three days; the colony surface was flat with a velvety texture; conidia were dense, forming a deep conidial layer; the conidial layer moved when the petri dish was tapped; the edge area of ​​the colony was white within a 12 mm wide region, turning pale green, and becoming dark green as it moved towards the center of the colony; no exudate was observed, and there was no obvious odor. Figure 2 As shown.

[0085] Combining morphological characteristics and molecular identification, a phylogenetic tree was constructed using the maximum likelihood (ML) method. Specifically, the internal transcriptional spacer (ITS) region of rRNA and the RNA polymerase II subunit B2 (rpb2) sequence were analyzed (mycelial samples were sent to Qingke Biotechnology Co., Ltd. for extraction, amplification, and sequencing). The sequencing results were input into GenBank (www.ncbi.nlm.nih.gov), and the phylogenetic tree was constructed using the maximum likelihood (ML) method based on homologous sequences in GenBank. The results are as follows: Figure 1 As shown, where Figure 1 The scale bar is 0.050.

[0086] Depend on Figure 1 The results show that the endophytic fungus MWTG-03 has a 99% similarity to Penicillium oxalicum in NCBI, thus confirming that the endophytic fungus isolated in this invention is a Penicillium endophytic fungus MWTG-03 (Penicillium oxalicum), which was named and biologically deposited with the accession number CCTCC M 20231220.

[0087] Example 2

[0088] The preparation method of dienepyrroloxamic acid bacteriocin is as follows:

[0089] 1. Activation of the strain: The Penicillium endophytic fungus isolated in Example 1 was inoculated onto PDA slant medium, cultured at 28°C for 5 days, and then stored in a refrigerator at 4°C for later use.

[0090] 2. Preparation of fermentation medium: Take washed potatoes, cut them into potato pieces with a diameter of 1cm and place them in tissue culture bottles (80g / bottle). After capping the tissue culture bottles, sterilize them at 120℃ for 30min and cool them to obtain the fermentation medium.

[0091] 3. The activated Penicillium endophytic fungi from step 1 were inoculated into the fermentation medium prepared in step 2 at an inoculation rate of 1 wt.%, covered, and cultured at 28°C for 30 days to obtain Penicillium fungal ferment.

[0092] 4. Mix 20 kg of the fermentation product obtained in step 3 with 10 L of ethyl acetate, sonicate at 40-53 kHz for 60 min, filter, and concentrate the filtrate under reduced pressure until there is no alcohol odor to obtain 46 g of Penicillium oxalate fermentation crude extract.

[0093] 5. Dissolve 46g of Penicillium oxalate fermentation crude extract in 15mL of dichloromethane-methanol solution with a volume ratio of 2:1, mix with 40g of 200-mesh silica gel, concentrate under reduced pressure to remove solvent, and pack into a column.

[0094] Gradient elution was performed sequentially with dichloromethane-methanol solutions at volume ratios of 100:0, 100:1, 50:1, 30:1, 10:1, and 3:1.

[0095] The eluent was concentrated and evaporated to dryness using a 10:1 dichloromethane-methanol solution. The eluent was then eluted by gel column chromatography using methanol as the solvent, and the eluent was concentrated.

[0096] The concentrated sample was tested using methanol as solvent and a preparative high-performance liquid chromatograph with a C18 column. The mobile phase was methanol and water, with a volume ratio of methanol to water of 6:4. The detection wavelength was 210 or 254 nm. The compound dienepyrroloxamic acid bacteriocin (5.2 mg) was prepared.

[0097] Example 3

[0098] The structural identification of the compound dienepyrroloxamic acid bacteriocin prepared in Example 2 was performed as follows:

[0099] The compound dienepyrroloxamic acid bacteriocin prepared in Example 2 was structurally identified by 1D / 2D NMR (one-dimensional nuclear magnetic resonance spectroscopy and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). The results are as follows. Figure 4 As shown.

[0100] Dienepyrroloxamic acid bacteriocin is a black crystal. HR-ESI-MS analysis showed that the quasi-molecular ion peak of this compound was at 220.0576 [M+Na]. + (Calculated as C9H) 11 NO4Na + (220.0586), indicating that its molecular formula is C 28 H 46 O4 (m / z 197.0688), with an unsaturation degree of 5.

[0101] Infrared spectroscopy (IR) showed the absorption of hydroxyl groups. Furthermore, 13 CNMR spectrum ( Figure 6 The presence of one methyl group (δ) was observed. C 20.8), 2 methylene (δ C 27.3, 66.4), 2 methines (δ C 116.5, 122.3), and 4 quaternary carbons (δ C 122.9, 124.0, 164.6, 173.0). From HSQC spectra ( Figure 8 The chemical shift δ of H and C associated with dienepyrrolopyridine can be obtained as shown in Table 1. Figures 5-6 :

[0102] Table 1 Dienepyrrole oxadiazine 1 H (400MHz) and 13 C(100MHz) NMR data

[0103]

[0104] The above data indicate that this compound possesses a 4-ethyl-2-methylpyrrole skeleton. The C-10 and C-7 chemical shifts are clearly downward shifts, suggesting they are bonded to oxygen atoms. HMBC correlations are obtained through H-7 / C-9 and C-6; H-5 / C-7. Figure 9 The molecular formula and its structure indicate that the acetoxy group is attached to C-7 via an oxygen atom, and the carboxylic acid carbon is attached to C-2. The planar structure is further... 1 H- 1 H COSY experiment ( Figure 7 ) and NOESY experiment ( Figure 10 This was confirmed. Finally, X-ray single-crystal diffraction experiments were used to further verify and determine the planar structure of the compound, as shown below. Figure 3 As shown, it was named dienepyrrolidin.

[0105] Example 4

[0106] The anti-inflammatory activity of dienepyrroloxamic acid bacteriocin was verified by the following steps:

[0107] Anti-inflammatory activity:

[0108] Nitric oxide (NO) has a wide range of important biological regulatory functions, playing a crucial role in inflammation, tumors, and the cardiovascular system. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., they generate large amounts of induced NO synthase (iNOS), producing NO to initiate an immune response. Therefore, inhibiting NO production is a direct indicator of the anti-inflammatory activity of compounds.

[0109] (1) Logarithmic growth phase mouse monocytes RAW264.7 (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were collected and stimulated with 1 μg / mL LPS. Simultaneously, dienepyrrolizoxalic acid prepared in Example 2 was added for treatment. The final concentrations of dienepyrrolizoxalic acid were 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM, with three replicates for each gradient. The cells were cultured at 25°C. A drug-free group and an L-NMMA (total NOS inhibitor) positive drug group were treated using the same steps as described above, serving as controls.

[0110] (2) After overnight (10 h) cell culture, NO production in each group was measured at 570 nm. MTS was added to the culture medium obtained after overnight culture to detect cell viability and eliminate the toxic effects of the compound on cells. NO production inhibition rate (%) = (OD of drug-free group) 570nm -Drug group OD 570nm ) / Drug-free group OD 570nm ×100%; IC 50The IC50 (half-maximal inhibitory concentration) of the NO inhibitory activity of dienepyrroloxamic acid bacteriocin was calculated using the Reed & Muench method. 50 The value is: 28.14±1.58μM.

[0111] Calculations showed that in the screening test for the NO inhibitory activity of the compound dienepyrroloxamic acid (positive control: L-NMMA), dienepyrroloxamic acid exhibited significant inhibitory activity against nitric oxide (NO) formation, with a half-maximal inhibitory rate (IC50) of 100%. 50 The half-maximal inhibitory concentration (IC50) of the positive control L-NMMA against nitric oxide (NO) was 28.14 ± 1.58 μM. 50 The value was 38.14 ± 1.25 μM, as shown in Table 2. The inhibition rate of nitric oxide (NO) production by dienepyrrole oxalicylate was significantly stronger than that of the positive control L-NMMA (IC50). 50 =38.14±1.25μM).

[0112] Table 2 Screening of anti-inflammatory activity of dienepyrrolizidine oxalic acid (IC50) 50 )

[0113] sample Concentration (μM) NO generation inhibition rate (%) <![CDATA[IC 50 (μM)]]> L-NMMA* 50 56.28±0.98 38.14±1.25 oxalicumloid 50 68.08±0.64 28.14±1.58

[0114] The results above demonstrate that dienepyrroloxamic acid bacteriocin possesses excellent anti-inflammatory activity and is non-cytotoxic. Furthermore, the method for producing large quantities of dienepyrroloxamic acid bacteriocin based on microbial fermentation offers advantages such as mild culture conditions, simple and easy culture medium preparation, short fermentation time, and ease of large-scale production.

[0115] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A dienepyrroloxamic acid bacteriocin, with the structure shown in Formula I: Formula I.

2. The method for preparing dienepyrroloxamic acid bacteriocin according to claim 1, characterized in that, Includes the following steps: Activated Penicillium endophytic fungi ( Penicillium oxalicum MWTG-03 was inoculated into sterilized potatoes for fermentation culture to obtain the fermentation product of the Penicillium endophytic fungus MWTG-03; the fermentation culture temperature was 18~28℃ and the time was 28~32 days. The fermentation product of the Penicillium endophytic fungus MWTG-03 was mixed with a polar organic solvent and then subjected to ultrasonic extraction to obtain a polar organic solvent extract. The polar organic solvent extract was subjected to silica gel column chromatography, gel column chromatography, and high performance liquid chromatography in sequence to obtain the dienepyrroloxamic acid bacteriocin; The silica gel column chromatography includes: sequentially eluting with dichloromethane-methanol systems at volume ratios of 100:0, 100:1, 50:1, 30:1, 10:1 and 3:1, collecting the elution fraction of dichloromethane-methanol solution at a volume ratio of 10:1 to obtain the silica gel column chromatography solution; The eluent for the gel column chromatography separation is methanol; The mobile phase for high-performance liquid chromatography (HPLC) separation is a methanol-water system; the volume ratio of methanol to water in the methanol-water system is 6~9:4~5; the HPLC separation is a preparative HPLC separation; and the chromatographic column for HPLC separation is a C18 column. The preservation number of the Penicillium endophytic fungus MWTG-03 is CCTCC M 20231220.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the fermentation product of the Penicillium endophytic fungus MWTG-03 to the volume ratio of the polar organic solvent is (30~80) g: (50~150) mL; the ultrasonic extraction frequency is 40~53 kHz and the time is 30~60 min.

4. The use of dienepyrroloxamic acid bacteriocin according to claim 1 or the preparation method according to claim 2 or 3 in the preparation of anti-inflammatory drugs.

5. An endophytic fungus of the genus Penicillium ( Penicillium oxalicum The preservation number of the Penicillium endophytic fungus MWTG-03 is CCTCC M 20231220.

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