Process for preparing polyketides from the fungus genus campylopus and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of a polyketide compound from a fungus of the genus Arenarius. The polyketide compound can dose-dependently reduce the NO generation level in cells, significantly inhibit M1 phenotype polarization and promote the conversion of the M1 phenotype to the M2 phenotype, significantly down-regulate the expression of key inflammatory-related proteins (including IL-1beta, IL-6, TNF-alpha and COX-2) in cells, and inhibit the generation of NLRP3 inflammasome and caspase-1, revealing the potential anti-AD activity and indicating that the polyketide compound has a good application prospect in the preparation of anti-neuroinflammatory drugs. In addition, the polyketide compound can be prepared from a marine microorganism Arenarius fungus, and the preparation method is simple and the source is rich.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation method and application of polyketide compounds derived from the genus *Curvus*. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that commonly affects the elderly or pre-aging populations. Clinically, it is characterized by progressive cognitive decline and behavioral disturbances, and often leads to various complications, severely impacting patients' quality of life. Although the exact cause of AD remains unclear, its main pathological features include β-amyloid protein deposition, abnormal aggregation of tau protein in neurons, a decrease in the number of cholinergic neurons and their synapses in the cerebral cortex and hippocampus, and amyloid degeneration of cortical arteries.

[0003] Neuroinflammation is often dynamically involved in the pathological process of neurological diseases, making it a hot research topic in neurodegenerative diseases in recent years. Neuroinflammation can directly damage the normal function of neurons and synapses, accelerate neuronal apoptosis, and thus exacerbate the pathological process of Alzheimer's disease (AD). Microglia activation is a key pathological feature of neuroinflammation. After activation, they can transform into the pro-inflammatory M1 phenotype, releasing pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as neurotoxic factors such as reactive oxygen species (ROS) and nitric oxide (NO), thereby maintaining chronic neuroinflammation and ultimately causing damage to the nervous system. Based on this, intervention strategies targeting neuroinflammation have become an important direction in AD drug development.

[0004] The ocean, as the world's largest treasure trove of biological resources, is an important source of natural products and provides a valuable material foundation for drug development. Thanks to the unique metabolic pathways and mechanisms of marine organisms, marine natural products often possess novel structures and significant activity, exhibiting great potential for drug discovery. Therefore, identifying anti-neuroinflammatory active substances from the ocean that have preventative effects against Alzheimer's disease (AD) or can delay its progression is of great significance for the development of AD drugs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of existing anti-neuroinflammatory drugs and to provide a method for preparing polyketide compounds derived from Curvularia fungi and their applications.

[0006] The technical objective of this invention is achieved through the following technical solution:

[0007] This invention also provides a method for preparing polyketides derived from *Curvaria* fungi, wherein the polyketides are prepared by fermentation, separation, and purification of *Curvaria* fungi, and the *Curvaria* fungi are... Eutypellasp. F0219, the polyketide compound has the structure of Formula 1:

[0008] .

[0009] Furthermore, the above preparation method specifically includes the following steps:

[0010] S1. Fungi of the genus *Curvus* Eutypella sp. F0219 was activated and cultured on a large scale to obtain the fermentation product;

[0011] S2. The fermentation product obtained in step S1 is extracted by soaking in ethyl acetate, and the crude extract is extracted with ethyl acetate to obtain ethyl acetate extract;

[0012] S3. The ethyl acetate extract obtained in step S2 is subjected to normal phase silica gel column chromatography, with dichloromethane-methanol as the eluent for gradient elution, and the fraction with a dichloromethane-methanol volume ratio of 10:1 is collected.

[0013] S4. The fraction obtained in step S3 with a dichloromethane-methanol volume ratio of 10:1 was subjected to ODS (octadecylsilyl) column chromatography with methanol-water as the eluent for gradient elution to obtain a fraction with a methanol volume fraction of 30%.

[0014] S5. The component with a methanol volume fraction of 30% obtained in step S4 is separated and purified by high performance liquid chromatography to obtain a polyketide compound.

[0015] Furthermore, in step S2, the fermentation product obtained in step S1 is extracted with ethyl acetate 2-3 times, each time for 3-4 days. The extract is then concentrated under reduced pressure to obtain a crude extract.

[0016] Furthermore, in step S3, gradient elution is performed at a dichloromethane-methanol volume ratio (v / v) of 100:0 to 0:100. Preferably, the elution gradient is 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, and 0:1.

[0017] Furthermore, in step S4, gradient elution is performed sequentially according to methanol-water volume ratios (v / v) of 30:70, 45:55, 60:40, 75:25, 90:10, and 100:0.

[0018] Furthermore, in step S5, the mobile phase of the high-performance liquid chromatography is 10% acetonitrile / water, the flow rate is 1~3 mL / min, the detection wavelength is 210 nm, and the retention time is 36 minutes.

[0019] This invention provides the use of polyketides derived from *Cyclocarya* fungi in the preparation of anti-neuroinflammatory drugs, wherein the polyketides include compounds represented by Formula 1:

[0020] .

[0021] Furthermore, the polyketide compound also includes pharmaceutically acceptable salts, solvates, or stereoisomers of the compound represented by Formula 1.

[0022] Furthermore, the polyketide compound is prepared by fermentation, separation, and purification of the *Curvularia* fungus, wherein the *Curvularia* fungus is... Eutypella sp. F0219.

[0023] Furthermore, the polyketide compounds can dose-dependently reduce the level of nitric oxide (NO) production in cells, significantly inhibit M1 phenotype polarization and promote its conversion to M2 phenotype, significantly downregulate the expression of key intracellular inflammation-related proteins (including IL-1β, IL-6, TNF-α and COX-2), and inhibit the expression of NOD-like receptor heat protein domain-associated protein 3 (NLRP3) inflammasome and cysteine-aspartate specific proteolytic enzyme-1 (Caspase-1).

[0024] This invention provides a method for preparing polyketides derived from the genus *Curvularia* and their applications. These polyketides can be prepared through fermentation, separation, and purification using marine microorganisms of the genus *Curvularia*. The preparation method is simple, the source is abundant, and it is conducive to industrial-scale production. Experiments have confirmed that the polyketides can reduce nitric oxide (NO) production levels in BV2 cells in a dose-dependent manner. The polyketides can also promote the conversion of LPS-induced BV2 cells from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. Simultaneously, they can inhibit the expression of NOD-like receptor heat protein domain-associated protein 3 (NLRP3) inflammasome and cysteine-aspartate specific proteolytic enzyme-1 (Caspase-1), revealing their potential anti-AD activity and indicating a promising application prospect in the preparation of anti-neuroinflammatory drugs. Attached Figure Description

[0025] Figure 1 Compound 1 of the present invention 1 H-NMR spectrum ( 1 H nuclear magnetic resonance spectrum).

[0026] Figure 2 Compound 1 of the present invention 13 C13 NMR spectrum (carbon-13 nuclear magnetic resonance spectrum).

[0027] Figure 3Compound 1 of the present invention 1 H- 1 H COSY spectrum (hydrogen-hydrogen isonuclear shift correlation spectrum).

[0028] Figure 4 The HSQC spectrum (heteronuclear single quantum correlation spectrum) of compound 1 of the present invention.

[0029] Figure 5 The HMBC spectrum (heteronuclear multibond correlation spectrum) of compound 1 of the present invention.

[0030] Figure 6 The NOESY spectrum (nuclear Overhaus effect spectrum) of compound 1 of the present invention.

[0031] Figure 7 HRESIMS (High-Resolution Electrospray Ionization Mass Spectrometry) spectrum of compound 1 of the present invention.

[0032] Figure 8 The results of the anti-neuroinflammatory activity test of compound 1 of the present invention are shown in Figure A, which shows the NO level, Figure B, which shows the cell viability, Figure C, which shows the results of Western blot analysis, Figure D, which shows the results of immunofluorescence staining, and Figure E, which shows the results of Western blot analysis. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Among them, the endophytic fungi used in fermentation Eutypella sp. F0219 separated from a certain sea area in China (GPS 114.6609) 21.5942 The seabed sediments at a depth of 75m belong to the genus *Curvularia*. Eutypella The fungi described are classified as *Cyclocarya*. Eutypella sp. F0219, provided by Hainan Normal University, is already published in existing literature (Yi Jiling, Shi Kangqi, Wu Binglin, et al. Marine-derived fungi). Eutypella Study on secondary metabolites of sp.F0219 [J]. Organic Chemistry, 2023, 43(1):4.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Example

[0037] I. Preparation of polyketide compounds shown in Formula 1

[0038] Specifically, the following steps are included:

[0039] S1. Activation of the strain: Extracting the preserved endophytic fungi. Eutypella After pre-culturing sp. F0219 in a constant temperature environment of 28℃ for 24 hours, it was transferred to a sterile PDA (potato dextrose agar) plate for activation and cultured in an incubator for 2-4 days.

[0040] S2. Seed liquid preparation: Take an endophytic fungus the size of an activated broad bean. Eutypella sp. F0219 cells were inoculated into sterile PDB (Potato Dextrose Broth) liquid medium and cultured in a shaker at 28°C for 2-3 days to obtain seed culture.

[0041] S3. Extract Preparation: The cultured seed liquid was transferred to sterile rice solid culture medium in a sterile laminar flow hood using a pipette, and fermented at a constant temperature of 28℃ for 28-31 days to obtain the fermentation product; the fermentation product was extracted by soaking in ethyl acetate solvent 2-3 times, each soaking for 3-4 days; the extract was concentrated under reduced pressure to obtain a crude extract; the crude extract was extracted with a water-ethyl acetate solvent with a volume ratio of 1:3, and the extraction was repeated 3-4 times to obtain an ethyl acetate extract;

[0042] S4. Column Chromatography Separation: The ethyl acetate extract was subjected to normal-phase silica gel column chromatography with dichloromethane-methanol solvents (100:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 0:1, v / v) for gradient elution. Approximately 1 L of fraction was collected each time, resulting in 331 fractions (Fr. 1 ~ Fr. 331). Similar fractions Fr. 113-140 (dichloromethane-methanol volume ratio 10:1) were combined by TLC and further analyzed using ODS column chromatography with methanol:water (v / v) = 30:70, 45:55, 60:40, 75:25, 90:10, 100:0 for gradient elution, yielding 96 fractions (Fr. 113-140-1 ~ Fr. 331). 113-140-96).

[0043] S5. Preparation of monomeric compounds: Fraction Fr. 113-140-5 (methanol:water = 30:70) was purified by high-performance liquid chromatography (HPLC) elution with a mobile phase of 12% acetonitrile / water, a flow rate of 3 mL / min, a detection wavelength of 210 nm, and a retention time of 29 min, yielding Fr. 113-140-5-1 ~ Fr. 113-140-5-6. Fraction Fr. 113-140-5-5 was further purified by HPLC elution with a mobile phase of 10% acetonitrile / water, a flow rate of 3 mL / min, a detection wavelength of 210 nm, and a retention time of 36 min, yielding compound 1.

[0044]

[0045] II. Structural confirmation of compound 1

[0046] The resulting compound 1 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 For H COSY, HSQC, HMBC, NOESY, and HRESIMS spectra, please refer to [link / reference]. Figures 1-7 The NMR data (400 MHz, δ in ppm, J in Hz) are shown in Table 1. The results show that the series of spectral data and NMR data of the obtained compound 1 correspond one-to-one with its structural formula, confirming that its structure is the structure of formula 1.

[0047] Table 1. NMR data of compound 1

[0048]

[0049] III. Anti-neuroinflammatory activity test of polyketide compound 1

[0050] 1. Cell Culture

[0051] Microglia (BV2) were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% streptomycin and penicillin, and passaged every two days.

[0052] 2. NO detection in LPS-induced BV2 microglia

[0053] BV2 microglia were seeded into 96-well plates. Once the cells adhered and reached approximately 50% confluence, LPS (1 μg / mL) was added for induction, followed by treatment with compound 1 (20 μM). After 24 hours of incubation, 50 μL of culture supernatant was transferred from each well to a new 96-well plate, and the nitrite content in the supernatant was immediately measured using a Griess reagent kit, serving as an indicator of NO content.

[0054] 3. Western Blot Analysis

[0055] BV2 cells were seeded in 6-well plates (2 mL / well) and incubated for 24 hours. Then, the cells were divided into five groups according to the treatment methods shown in Table 2: blank control, LPS-induced group (1 μg / mL), LPS + compound 1 (10 μM), LPS + compound 1 (20 μM), and LPS + compound 1 (40 μM), and cultured for another 24 hours. After culture, proteins were extracted using 1× RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis buffer) containing a protease inhibitor. After protein quantification, an equal volume of lysis buffer (20 μg / lane) was used for 10% SDS-PAGE electrophoresis (sodium dodecyl sulfonate-polyacrylamide gel electrophoresis) and transferred to a 0.22 μm PVDF membrane.

[0056] Table 2. Experimental Groups

[0057]

[0058] The membrane was blocked for 1 hour at room temperature using TBST blocking buffer containing 5% skim milk powder. Subsequently, it was blocked at 4°C with primary antibodies iNOS (inducible nitric oxide synthase, Abcam, catalog number ab178945), COX-2 (cyclooxygenase-2, Affinity, catalog number AF7003), IL-1β (interleukin-1β, CST (Cell Signaling Technology, catalog number 26048-1-AP), and IL-6 (interleukin-6, Affinity). The primary antibody was incubated overnight with HRP-labeled secondary antibody (catalog number DF6087), TNF-α (tumor necrosis factor-α, Affinity, catalog number AF7014), ARG-1 (human arginase 1, Proteintech, catalog number 66129), GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Bioss, catalog number bs-41373R), Caspase-1 (cysteine-aspartate specific proteolytic enzyme-1, Affinity, catalog number AF5418), and NLRP3 (NOD-like receptor thermoprotein domain-associated protein 3, Affinity, catalog number DF7438). After incubation, unbound primary antibody was washed away, and the mixture was then incubated with HRP-labeled secondary antibody at room temperature for 45 minutes. Finally, the target protein bands were visualized using an ECL kit (Millipore, catalog number 6-201BP).

[0059] 4. Immunofluorescence experiment

[0060] BV2 cells were seeded in culture dishes and treated with drugs the following day. After treatment, the cells were fixed and blocked with 5% BSA (Servicebio, catalog number GC305010). Subsequently, they were incubated sequentially with primary antibodies against ARG1 (Proteintech, catalog number 66129) and iNOS (Proteintech, catalog number 66129), followed by incubation with corresponding fluorescently labeled secondary antibodies. Finally, images were acquired using a confocal microscope.

[0061] 5. Experimental Results:

[0062] The test results of the anti-neuroinflammatory activity of the polyketide compound 1 of this invention are as follows: Figure 8 As shown. The polyketide compound 1 of this invention can significantly inhibit NO production in BV2 cells and reduces intracellular NO production levels in a concentration-dependent manner (as shown). Figure 8 (See Figure A). Furthermore, the CCK8 assay kit (Biosharp, catalog number BS350B) showed that cell viability was greater than 94% in the range of 1.25 μM to 40 μM, indicating that compound 1 had no significant toxic side effects on BV2 cells and possessed good biocompatibility. Figure 8 (Figure B in the middle)

[0063] In Western blot analysis, as the concentration of compound 1 increased, the protein band of iNOS (M1 marker) gradually faded, while the band of ARG-1 (M2 marker) gradually deepened, indicating that compound 1 can significantly inhibit M1 polarization and promote its conversion to the M2 phenotype. Figure 8 (Figure C). Immunofluorescence staining results showed that as the concentration of compound 1 increased, the intensity of the red fluorescence (labeled with M1 marker iNOS) of the M1 marker gradually decreased, while the green fluorescence of the M2 marker ARG-1 increased accordingly. This phenomenon further corroborates that compound 1 can promote the LPS-induced conversion of BV2 cells from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Figure 8 (See Figure D in the original text). Furthermore, Western blot analysis showed that compound 1 downregulated the expression of several key inflammation-related proteins in a concentration-dependent manner, including IL-1β, IL-6, TNF-α, and COX-2, and that compound 1 inhibited the production of the NLRP3 inflammasome and caspase-1. Figure 8 (China E map).

[0064] In summary, the polyketide compound 1 of this invention not only has excellent biosafety, but also significantly inhibits LPS-induced cellular inflammatory responses at certain concentrations, exhibiting good anti-neuroinflammatory effects. The effective amount of the compound of this invention can be used to prepare drugs for the prevention or treatment of diseases caused by neuroinflammation, to alleviate the symptoms of diseases caused by neuroinflammation, or to delay the development or onset of diseases caused by novel viruses, and has broad prospects for pharmaceutical research and development and application.

[0065] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing polyketide compounds derived from *Curvaria* fungi, characterized in that, The polyketide compound was prepared by fermentation, separation, and purification of the *Curvularia* fungus. The *Curvularia* fungus is... Eutypella sp.F0219, the polyketide compound is the compound shown in Formula 1: 。 2. The preparation method according to claim 1, characterized in that, The preparation method specifically includes the following steps: S1. Fungi of the genus *Curvus* Eutypella sp. F0219 was activated and cultured on a large scale to obtain the fermentation product; S2. The fermentation product obtained in step S1 is extracted by soaking in ethyl acetate, and the crude extract is extracted with ethyl acetate to obtain ethyl acetate extract; S3. The ethyl acetate extract obtained in step S2 is subjected to normal phase silica gel column chromatography, with dichloromethane-methanol as the eluent for gradient elution, and the fraction with a dichloromethane-methanol volume ratio of 10:1 is collected. S4. The fraction obtained in step S3 with a dichloromethane-methanol volume ratio of 10:1 was subjected to ODS column chromatography, and gradient elution was performed using methanol-water as the eluent to obtain a fraction with a methanol volume fraction of 30%. S5. The component with a methanol volume fraction of 30% obtained in step S4 is separated and purified by high performance liquid chromatography to obtain a polyketide compound.

3. The preparation method according to claim 2, characterized in that, In step S2, the fermentation product obtained in step S1 is extracted with ethyl acetate 2-3 times, each time for 3-4 days. The extract is then concentrated under reduced pressure to obtain a crude extract.

4. The preparation method according to claim 2, characterized in that, In step S3, gradient elution is performed at a dichloromethane-methanol volume ratio of 100:0 to 0:

100.

5. The preparation method according to claim 2, characterized in that, In step S4, gradient elution is performed sequentially according to methanol-water volume ratios of 30:70, 45:55, 60:40, 75:25, 90:10, and 100:

0.

6. The preparation method according to claim 2, characterized in that, In step S5, the mobile phase of the high-performance liquid chromatography (HPLC) is 10% acetonitrile / water, the flow rate is 1~3 mL / min, the detection wavelength is 210 nm, and the retention time is 36 minutes.

7. The application of polyketides derived from *Curvaria* fungi in the preparation of anti-neuroinflammatory drugs, characterized in that, The polyketide compounds include those shown in Formula 1: 。 8. The application according to claim 7, characterized in that, The polyketide compounds also include pharmaceutically acceptable salts, solvates, or stereoisomers of the compounds shown in Formula 1.

9. The application according to claim 7, characterized in that, The polyketide compound was prepared by fermentation, separation, and purification of the *Curvularia* fungus. The *Curvularia* fungus is... Eutypella sp. F0219.

10. The application according to claim 7, characterized in that, The polyketide compounds can reduce the level of nitric oxide production in cells in a dose-dependent manner, significantly inhibit M1 phenotype polarization and promote its conversion to M2 phenotype, significantly downregulate the expression of key intracellular inflammation-related proteins, and inhibit the expression of NOD-like receptor thermoprotein domain-associated protein 3 inflammasome and cysteine-aspartate specific proteolytic enzyme-1.

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