Polyketone compound as well as separation method and application thereof

Abstract

The invention relates to the technical field of compounds, and particularly discloses a polyketone compound and a separation method and application thereof.The molecular formula of the compound is C36H42O10, and the compound has a unique eight-membered ring skeleton structure and is named as neocitrinol D; the citrinin dimer has a structural formula shown as a formula (I): imgabs0 #. Experiments prove that the citrinin dimer has relatively better anti-inflammatory activity compared with the known citrinin dimer (compounds 2-5) separated from a nigrospora oryzae strain, is high in safety and has an extremely good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of new compounds, and in particular to a polyketide compound, a separation method thereof and an application thereof. Background Art

[0002] Nigrospora oryzae Nigrospora oryzae belongs to Deuteromycotina, Hyphomycetes, Hyphomycetales, Dematiaceae, Nigrospora, and is particularly common in tropical regions. It can infect plants such as sorghum, corn, wheat, Kentucky bluegrass, Ficus religiosa, and cotton. Nigrospora oryzae can produce a large number of bioactive metabolites. Related research shows that solanapyrones A and B isolated from the fermentation medium of Nigrospora oryzae, as well as an undescribed pyrone (solanapyrone U), can induce bone marrow mesenchymal stem cells (bMSCs) to secrete nerve growth factor (NGF). Two novel solanapyrone analogues, solanapyrones N and solanapyrones O, found in the fermentation culture of the Nigrospora strain YB-141, have mild antifungal activity. A novel alkaloid, nigrosporine B, identified from the marine fungus Nigrospora oryzae SYSU-MS0024, has moderate inhibitory activity against acetylcholinesterase (AChE), and its half-maximal inhibitory concentration (IC 50 ) value is 103.7 μmol / L. However, there are still few reports on the application of Nigrospora oryzae metabolites in anti-inflammatory aspects.

[0003] Based on this, the present invention aims to isolate a new compound with excellent anti-inflammatory effects from Nigrospora oryzae metabolites to enrich the chemical diversity and biological activity of Nigrospora oryzae. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention provides a polyketide compound, a separation method thereof and an application thereof. The molecular formula of the polyketide compound is C 36 H 42 O 10 , named neocrinonol D. In addition, the present invention proves through experiments that this compound has relatively better anti-inflammatory activity compared to the known citrinin dimer (compound 2-5) isolated from Nigrospora oryzae strains, and its inhibitory effect on NO production is higher than that of the conventional anti-inflammatory agent Sappanone A, and it has extremely high application prospects in the preparation of anti-inflammatory products.

[0005] In the first aspect of the present invention, there is provided a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, .

[0006] The compound according to the embodiment of the present invention has at least the following beneficial effects: The present invention proves through experiments that this compound has relatively better anti-inflammatory activity compared with the known citrinin dimer (Compound 2-5) isolated from the strain of Nigrospora oryzae, and can significantly inhibit the generation of NO. Its inhibitory effect is even higher than that of the conventional anti-inflammatory agent Sappanone A. The IC 50 value is 3.934 μM. In addition, within the effective dose range of this compound (such as at 10 μM), it has almost no toxic effect on cells, indicating its high safety and extremely high application prospects in the preparation of anti-inflammatory products.

[0007] In some embodiments of the present invention, the molecular formula of the compound is C 36 H 42 O 10 , and the conformation is 2 R ,3 R ,4 S ,5 R ,9 S ,2' R ,3' S ,10' R ,2'' R ,3'' S ,10'' S .

[0008] In some embodiments of the present invention, the structural formula of the compound is as follows: .

[0009] In some embodiments of the present invention, the pharmaceutically acceptable salt refers to the salt of the free acid or base of the compound referred to herein, which is non-toxic, biocompatible or otherwise biologically suitable for administration to a subject. Generally see, S.M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those salts that are pharmacologically effective and suitable for contact with the tissues of the subject without excessive toxicity, irritation or allergic reaction. The compounds described herein can have groups with sufficient acidity, groups with sufficient basicity, both types of functional groups, or more than one functional group of each type, and thus react with many inorganic or organic bases as well as inorganic and organic acids to form pharmaceutically acceptable salts.

[0010] For a compound containing a basic group (such as an amine) as described herein, pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, etc.) or an organic acid (such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, oxaloacetic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranosiduronic acid (such as glucuronic acid or galacturonic acid), α-hydroxy acid (such as mandelic acid, citric acid or tartaric acid), amino acid (such as aspartic acid or glutamic acid), aromatic acid (such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid or cinnamic acid), sulfonic acid (such as laurylsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid or ethanesulfonic acid)), or any compatible mixture of acids (such as those given as examples herein), and any other acid and its mixture regarded as equivalents or acceptable substitutes according to the ordinary skill level in the art.

[0011] For a compound containing an acidic group (such as a carboxylic acid group) as described herein, base addition salts can be prepared by any suitable method available in the art, for example, by treating such a compound with a sufficient amount of the desired base (soda ash or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, ammonium, zinc or magnesium salts, or other metal salts; organic amino salts, such as alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts.

[0012] In some preferred embodiments of the present invention, the pharmaceutically acceptable salt is selected from any one of disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprinate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

[0013] In a second aspect of the present invention, there is provided a method for preparing a compound as described in the first aspect of the present invention, which comprises: synthesizing by chemical synthesis method, or isolating from the metabolites of the strain of Nigrospora oryzae ).

[0014] In some embodiments of the present invention, the Nigrospora oryzae strain was purchased from Beina Biology, with the number BNCC150488.

[0015] In some embodiments of the present invention, the separation from the metabolites of the Nigrospora oryzae strain includes: S1. Inoculate the Nigrospora oryzae strain into a culture medium for fermentation to obtain a fermentation product; S2. Extract and concentrate the fermentation product to obtain a crude extract; S3. Perform chromatographic separation on the crude extract, and then obtain the product after purification.

[0016] In some embodiments of the present invention, the culture medium is a rice culture medium.

[0017] In some embodiments of the present invention, the rice culture medium contains rice, yeast extract, and glucose.

[0018] In some embodiments of the present invention, the mass ratio of the rice, the yeast extract, and the glucose is 50:0.1 - 1:0.1 - 1. For example, it can be 50:0.25:0.25.

[0019] In some embodiments of the present invention, the fermentation temperature is 24 - 30 °C. For example, it can be 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C, etc.

[0020] In some embodiments of the present invention, the fermentation time is 20 - 40 days. For example, it can be 20 days, 22 days, 24 days, 25 days, 26 days, 28 days, 30 days, 32 days, 34 days, 36 days, 38 days, or 40 days, etc.

[0021] In some embodiments of the present invention, the chromatographic separation includes MCI column chromatographic separation.

[0022] In some embodiments of the present invention, the purification includes high - performance liquid chromatography purification.

[0023] In the third aspect of the present invention, there is provided the use of the compound described in the first aspect of the present invention or its pharmaceutically acceptable salt in the preparation of an anti - inflammatory product.

[0024] In some embodiments of the present invention, the product includes a drug or a functional food.

[0025] In the fourth aspect of the present invention, there is provided an anti - inflammatory drug, the active ingredient of which comprises the compound described in the first aspect of the present invention or its pharmaceutically acceptable salt.

[0026] In some embodiments of the present invention, the anti-inflammatory drug further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient is a conventional excipient in the pharmaceutical field.

[0027] In some embodiments of the present invention, the pharmaceutically acceptable excipient includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, a sweetening agent, and a flavoring agent.

[0028] In some embodiments of the present invention, the excipient includes water.

[0029] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0030] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, an alginate, gelatin, and polyvinylpyrrolidone.

[0031] In some embodiments of the present invention, the wetting agent includes glycerol.

[0032] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0033] In some embodiments of the present invention, the absorption promoter includes a quaternary ammonium compound.

[0034] In some embodiments of the present invention, the surfactant includes cetyl alcohol.

[0035] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and saponite clay.

[0036] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0037] In some embodiments of the present invention, the dosage form of the anti-inflammatory drug is various conventional dosage forms in the art.

[0038] In some embodiments of the present invention, the dosage form of the anti-inflammatory drug is in the form of a solid, semi-solid, or liquid, and can be an aqueous solution, a non-aqueous solution, or a suspension.

[0039] In some embodiments of the present invention, the dosage form of the anti-inflammatory drug is a tablet, a capsule, a soft capsule, a granule, a pill, an oral liquid, a dry suspension, a dropping pill, a dry extract, an injection, or an infusion.

[0040] In some embodiments of the present invention, the administration mode of the anti-inflammatory drug can be a conventional administration mode in the art, including but not limited to injection administration or oral administration.

[0041] In some embodiments of the present invention, the injection administration can be via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, subcutaneous injection or the like.

[0042] Other features and advantages of the present invention will be described in the subsequent specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present invention will be further described below in conjunction with the drawings and embodiments, wherein: Figure 1 are the 1 H (600 MHz) NMR data of Compound 1 of the present invention.

[0044] Figure 2 are the 13 C (150 MHz) NMR data of Compound 1 of the present invention.

[0045] Figure 3 are the important correlations of HMBC and NOESY of Compound 1 of the present invention.

[0046] Figure 4 are the ECD comparisons of (2 R ,3 R ,4 S ,5 R ,9 S ,2' R ,3' S ,10' R ,2'' R ,3'' S ,10'' S )-1 and (2 S ,3 S ,4 R ,5 S ,9 R ,2' S ,3' R ,10' S ,2'' S ,3'' R ,10'' R )-1 of the present invention at the B3LYP / 6-311G level in methanol.

[0047] Figure 5The cytotoxicity of Compound 1 of the present invention and its inhibitory effect on NO production. In A, the cell viability detection results of RAW 264.7 cells treated with 10 μM Compound 1 for 24 h are shown, and the data are expressed as mean ± SEM. In B, the statistical results of NO production are shown, **** P <0.0001 vs. LPS group; #### P <0.0001 vs. blank group.

[0048] Figure 6 The inhibitory effect of Compounds 2-5 of the present invention on NO production, **** P <0.0001 vs. LPS.

[0049] Figure 7 The inhibitory activity of Compound 1 of the present invention on NO production in LPS-induced RAW 264.7 cells. Detailed implementation manners

[0050] The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with the embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0051] The terms "preferably", "more preferably", etc. in the present invention refer to the embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0052] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to an integer, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0053] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the related listed items.

[0054] In the description of the present invention, the description with reference to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0055] For those not specifying specific conditions in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0056] Example 1: Strain Cultivation and Metabolite Separation 1. Strain Obtaining The strain used in this example is Nigrospora oryzae ( Nigrospora oryzae ), which is purchased from Beina Biology, with the number BNCC150488. Using glycerol with a final concentration of 20% as a cryoprotectant, it is stored in an environment of -80 °C. This strain sample is stored in the Integrated Chinese and Western Medicine Syndrome Laboratory of Southern Medical University, China, for standby.

[0057] 2. Strain Fermentation In a laminar flow hood, the mycelium of the above Nigrospora oryzae is inoculated into a rice medium and left to ferment statically at 26 °C for 30 days to obtain a fermentation product for standby.

[0058] The preparation method of the rice medium is as follows: Weigh 50 g of rice, 0.25 g of yeast extract, and 0.25 g of glucose, then add water to make up to 75 mL. After mixing evenly, it is sterilized at high temperature and then cooled for standby.

[0059] 3. Metabolite Separation (1) Obtaining the Crude Extract: The above fermentation product is extracted with 80% methanol and then extracted three times with ethyl acetate. 122.8 grams of crude extract is obtained by vacuum concentration.

[0060] (2) Chromatographic Separation: The above-mentioned crude extract was separated by MCI column chromatography. The filled MCI column was rinsed thoroughly with 100% methanol solvent, and the column was equilibrated to methanol: water (20:80). After mixing the ethyl acetate extract with the sample, it was loaded onto the column. Through MCI column chromatography, methanol-water was used as the mobile phase (20:80, 2 BV; 30:70, 2 BV; 40:60, 2 BV; 45:55, 2 BV; 50:50, 2 BV; 55:45, 2 BV; 60:40, 2 BV; 65:35, 2 BV; 70:30, 4 BV; 75:25, 4 BV; 80:20, 2 BV; 85:15, 2 BV; 90:10, 2 BV; 100:0, 3 BV) for gradient elution. Combining with the TLC results, a total of nine fractions (Fr.1-Fr.9) were obtained by segmenting.

[0061] Take the Fr.4 fraction (23.1 g) and use MCI column chromatography (the filled MCI column was rinsed thoroughly with 100% methanol solvent, and the column was equilibrated to methanol: water (30:70). After mixing the ethyl acetate extract with the sample, it was loaded onto the column for further separation). Using methanol-water (30%-60%) as the gradient eluent, eight fractions (Fr.4.1-Fr.4.8) were obtained.

[0062] Among them, the Fr.4.7 fraction (0.9 g) was further separated by Sephadex LH-20 (methanol) column chromatography to obtain six parts (Fr.4.7.1-Fr.4.7.6). The Fr.4.7.6 fraction was separated by semi-preparative high performance liquid chromatography (HPLC) with the mobile phase of acetonitrile-water (74%) to obtain Compound 1 (11.5 mg, retention time t R = 16 min). The Fr.4.7.2 fraction was separated by preparative HPLC with the mobile phase of methanol-water (83%) to obtain Compound 3 (5.8 mg, retention time t R = 17 min). The Fr.4.8 fraction (0.53 g) was purified by semi-preparative HPLC with the mobile phase of methanol-water (100%) to obtain Compound 2 (2.7 mg, retention time t R = 18 min).

[0063] After identification, the chemical structural formula of this Compound 2 is as follows: .

[0064] After identification, the chemical structural formula of this Compound 3 is as follows: .

[0065] The fraction Fr.6 (9.8 g) was separated by reversed-phase C18 column chromatography with a mobile phase of methanol-water (62% - 85%) to give four fractions (Fr.6.1 - Fr.6.4). Among them, the fraction Fr.6.4 (2.2 g) was further separated by Sephadex LH-20 (100% methanol) column chromatography to give four fractions (Fr.6.4.1 - Fr.6.4.4). The fraction Fr.6.4.1 (0.38 g) was separated by preparative HPLC with a mobile phase of methanol-water (97%, containing 0.1% formic acid) to give four fractions (Fr.6.4.1.1 - Fr.6.4.1.4). Finally, the fraction Fr.6.4.1.4 was purified by semi-preparative HPLC with a mobile phase of acetonitrile-water (92%) to give Compound 4 (2.4 mg, retention time t R = 11 min).

[0066] The chemical structure of Compound 4 was identified as follows: .

[0067] The fraction Fr.7 (14.0 g) was separated by reversed-phase C18 column chromatography with a gradient eluent of water-methanol (20% - 50%) to give seven components (Fr.7.1 - Fr.7.7). The fraction Fr.7.3 (2.8 g) was first purified by Sephadex LH-20 (methanol) column chromatography and then separated by semi-preparative HPLC with a mobile phase of acetonitrile-water (75%) to give Compound 5 (5.8 mg, retention time t R = 20.5 min).

[0068] The chemical structure of Compound 5 was identified as follows: .

[0069] Example 2: Structure Analysis of Compound 1 In this example, the structure of the above-separated Compound 1 was analyzed, which specifically includes the following content.

[0070] 1. High-Resolution Mass Spectrometry Analysis The above-separated Compound 1 (yellow powder) was subjected to high-resolution mass spectrometry (HRESIMS) analysis. The result showed that its ion peak was m / z 635.2825, from which its molecular formula was deduced to be C 36 H 42 O 10 , indicating that this compound has 16 degrees of unsaturation.

[0071] 2. Nuclear Magnetic Resonance Analysis The separated compound 1 was subjected to nuclear magnetic resonance analysis. The 1 H (600 MHz) and 13 C(150 MHz) NMR data are Figure 1 and Figure 2 shown as follows.

[0072] Analysis of the 1 H NMR hydrogen spectrum data revealed the presence of 14 sp 2 hybridized olefinic carbon atoms and 1 ketone carbonyl carbon atom ( δ C 199.3), which accounted for 8 degrees of unsaturation. The hydrogen spectrum data of compound 1 also revealed the presence of multiple methyl groups, including four singlet methyl groups with chemical shifts of δ H 3.56 (s, H3-14''), δ H 2.15 (overlap H3-13',H3-13''), δ H 1.75 (s, H3-12); six doublet methyl groups with chemical shifts of δ H 1.08 (d, J = 6.4 H z , H3-11), δ H 1.20 (d, J = 6.0 H z , H3-10), δ H 1.33 (d, J = 6.3 H z , H3-11'), δ H 1.26 (d, J = 7.1H z , H3-12'), δ H 1.36 (d, J = 6.3 H z , H3-11''), δ H 1.23 (d, J = 6.7 H z , H3-12'').

[0073] Secondly, 13 the C NMR carbon spectrum data showed a total of 36 carbon signals. Based on 1 the H NMR and 1313C NMR NMR data comparison shows that the heptacyclic B-H skeleton unit in Compound 1 is almost identical to that of the known neotriterpenoid A. However, there are significant differences in the A-ring unit between Compound 1 and neotriterpenoid A.

[0074] Further identification was carried out by two-dimensional nuclear magnetic resonance (2D NMR), and the results are as Figure 3 shown. In the HMBC spectrum, it can be observed that H3-12'' is correlated with C-2'', C-3'', C-4'', H-10'' is correlated with C-4'' and C-8'', H3-11'' is correlated with C-2'' and C-3'', H-2'' is correlated with C-10'', and H3-14'' is correlated with C-10''. These evidences reveal the existence of a six-membered ring unit. Further, through the analysis of NOESY correlation signals and biosynthetic pathways, the structure of the B-H ring part of Compound 1 is consistent with that of the known compound neotriterpenoid A. Therefore, the relative configuration of this part can be inferred to be 2 R *,3 R *,4 S *,5 R *,9 S *,2' R *,3' S *,10' R *. In the NOESY spectrum, the correlation between H3-12'' and H-2'', and the correlation between H3-11'' and H-3'' indicate that the spatial orientations of H-2'' and H-3'' are opposite (as Figure 3 shown), and the correlations between H-10'' and H3-11'', and between H3-11 and H3-12'' indicate that H-10'' is of β configuration.

[0075] 3. Circular dichroism (CD) test and quantum chemical calculation To further determine the absolute configuration of Compound 1, circular dichroism (CD) test and quantum chemical calculation were carried out in this example. Specifically, conformational optimization and calculation were performed on (2 R *,3 R *,4 S *,5 R *,9 S *,2' R *,3' S *,10' R *,2'' R *,3'' S *,10'' S *)-1 using Gaussian16 software.

[0076] The results are asFigure 4 As shown, showing (2 R , 3 R , 4 S , 5 R , 9 S , 2' R , 3' S , 10' R , 2'' R , 3'' S , 10'' S )-1 is in good agreement with the experimental value of compound 1. Therefore, compound 1 was identified as 2 R , 3 R , 4 S , 5 R , 9 S , 2' R , 3' S , 10' R , 2'' R , 3'' S , 10'' S , and named neocrinodione D.

[0077] The chemical structure of neocrinodione D (compound 1) is as follows: .

[0078] Detection example: Anti-inflammatory activity assay In this detection example, the anti-inflammatory activities of the above compounds 1-5 were detected, and the specific method is as follows.

[0079] 1. Cell culture: RAW264.7 cells were inoculated into complete DMEM medium (containing 10% fetal bovine serum (FBS), 1% of 100 units / mL penicillin and 100 mg / mL streptomycin), and cultured in an incubator at 37°C with 5% carbon dioxide for later use.

[0080] Then the cells were inoculated into a 96-well plate at a density of 5×10 5 cells / mL and cultured overnight. After the cell density reached 80%, lipopolysaccharide (LPS, final concentration 1 μg / mL) was added to induce RAW 264.7 cells for 24 hours to construct an inflammation model.

[0081] 2. Cell viability assay: RAW 264.7 cells (at a concentration of 5×10 5Cells / mL) were seeded into 96-well plates containing complete DMEM medium. After overnight incubation, the cells were treated with medium containing 10 μM compound 1 or 0.1% dimethyl sulfoxide (DMSO) for 24 h. Then, CCK-8 (produced by Biosharp, Beijing, China) was added to each well and incubated at 37 °C for 1 h. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (BioTek, USA).

[0082] 3. Bioactivity assay of nitric oxide production: The Griess method was used to determine the production of nitric oxide (NO) in RAW 264.7 cells to evaluate the anti-inflammatory effects of compounds 1-5. The specific procedure was as follows: RAW 264.7 cells were seeded into 96-well plates at a density of 5×10 5 cells per well and incubated overnight at 37 °C. Subsequently, the cells were treated with medium containing compounds 1-5 or 1% DMSO, and then stimulated with lipopolysaccharide (LPS, 1 μg / mL) for 24 h. Sappanone A (20 μM) was used as a positive control group.

[0083] Furthermore, to determine the half-maximal inhibitory concentration (IC 50 ) of compound 1, the cells were treated with compound 1 at concentrations of 10 μM, 5 μM, 2 μM, and 1 μM plus LPS (1 μg / mL) for 24 h. Finally, the content of NO in the supernatant was measured at a wavelength of 540 nm using a microplate reader with Griess reagent produced by Beyotime Biotechnology, Shanghai, China.

[0084] 4. Statistical analysis: Graphpad Prism 10.1.2 software was used to process the data. One-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test was used to determine statistical differences. All data were presented as means ± standard error of the mean (means ± SEM).

[0085] Figure 5 The cytotoxicity of compound 1 and its inhibitory effect on NO production are shown. Among them, A shows the results of cell viability detection after treatment with 10 μM compound 1 for 24 h on RAW 264.7 cells. The data are expressed as means ± SEM. B shows the NO production. The concentration of compound 1 is 10 μM, and the concentration of the positive control Sappanone A is 20 μM, **** P <0.0001 vs. LPS group; #### P<0.0001 vs. the blank group. The results showed that Compound 1 maintained good cell safety at low concentrations and exhibited significant anti-inflammatory activity, providing a key basis for its use as an anti-inflammatory candidate drug.

[0086] Figure 6 The inhibitory effects of Compounds 2-5 on NO production were demonstrated, where the concentrations of Compounds 2-5 were 20 μM, and Sappanone A (20 μM) was used as a positive control. **** P <0.0001 vs. LPS. The results showed that Compounds 2-5 did not exhibit anti-inflammatory activity.

[0087] Figure 7 The inhibitory activity of Compound 1 on NO production in LPS-induced RAW 264.7 cells (IC 50 value) was demonstrated. The results showed that its IC 50 value was 3.934 μM, and Compound 1 had good inhibitory activity on NO production.

[0088] In summary, the present invention provides a polyketide compound, its separation method and application. The present invention used Nigrospora oryzae ( Nigrospora oryzae ) fermented on a rice medium to isolate neoechinulin D (Compound 1) and four known citrinin dimers (Compounds 2-5). Structurally, Compound 1 has a novel 6 / 6 / 5 / 5 / 6 / 6 / 6 / 6 / 6 octacyclic carbon skeleton. Biologically, Compound 1 has a significant inhibitory effect on the production of NO, with an IC 50 value of 3.934 μM, and has very good development and application prospects.

[0089] The above has described the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, 。 2. A method for preparing the compound according to claim 1, characterized in that, comprising: Synthesized by chemical synthesis method or isolated from the metabolites of the strain Nigrospora oryzae Nigrospora oryzae ).

3. The preparation method according to claim 2, characterized in that, The strain of Nigrospora oryzae was purchased from Beina Biology, with the number BNCC150488.

4. The preparation method according to claim 2, wherein, The isolation from the metabolites of the Nigrospora oryzae strain includes: S1. Inoculating the Nigrospora oryzae strain into a culture medium for fermentation to obtain a fermentation product; S2. Extracting and concentrating the fermentation product to obtain a crude extract; S3. Performing chromatographic separation on the crude extract, and then purifying to obtain the product.

5. The preparation method according to claim 4, characterized in that, The culture medium is a rice culture medium.

6. The preparation method according to claim 4, characterized in that, The temperature of the fermentation is 24 - 30 °C; and / or, the time of the fermentation is 20 - 40 days.

7. The preparation method according to claim 4, characterized in that, The chromatographic separation includes MCI column chromatographic separation.

8. Use of the compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-inflammatory product.

9. An anti-inflammatory drug, characterized in that, The active ingredient contains the compound or a pharmaceutically acceptable salt thereof according to claim 1.

10. The anti-inflammatory drug according to claim 9, characterized in that, It further contains pharmaceutically acceptable excipients.

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