Mangrove forest sediment mud-derived fungus fermentation extract and preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, and specifically discloses a mangrove sediment mud-derived fungal fermentation extract, a preparation method and application thereof. Pseudallescheria angusta SYSU-M4 is inoculated into a liquid culture medium to obtain a spore liquid; the preservation number of the mangrove sediment mud-derived fungus is GDMCC No: 66263; (2) the spore liquid is inoculated into a solid culture medium to obtain a fermentation product; (3) the fermentation product is extracted by an extraction solvent, extracted by an extraction solvent, and separated by a separation solvent, and a component with a detection relative migration value of 0.4 in thin layer chromatography is collected, and the fermentation extract is obtained. The fermentation extract has obvious acetylcholinesterase and NO inhibitory activity, and has a good application prospect in the preparation of anti-inflammatory drugs and anti-inflammatory health products. The preparation method is simple in operation and suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a mangrove sediment mud-derived fungal fermentation extract and a preparation method and application thereof. BACKGROUND

[0002] With the continuous progress of society and the change of people's lifestyle, the incidence of nervous system diseases and inflammation-related diseases is gradually increasing, which has brought a great threat to people's health. Acetylcholinesterase plays an important role in the process of neural signal transmission, and its abnormal activity is closely related to various nervous system diseases such as Alzheimer's disease. As a defense mechanism of the body, excessive inflammation can cause tissue damage and disease progression, such as rheumatoid arthritis, inflammatory bowel disease, etc.

[0003] At present, drugs targeting acetylcholinesterase and inflammation are mainly divided into direct-acting drugs and indirect-acting drugs. Direct-acting drugs directly bind to acetylcholinesterase or inflammatory factors to inhibit their activity and rapidly relieve symptoms; indirect-acting drugs regulate the physiological processes of the body, such as regulating the metabolism of neurotransmitters or enhancing the body's anti-inflammatory ability, thereby achieving therapeutic effects.

[0004] Mangrove sediment mud is rich in various microorganisms, which have unique metabolic pathways and physiological characteristics and can produce secondary metabolites with antibacterial, antiviral, antitumor and other activities, and are an important source for developing new drugs and bioactive substances. At present, the research on mangrove sediment mud-derived strains focuses on the analysis of medicinal mechanisms, and various components of each part are explored, but the research on endophytic fungi in sediment mud is still blank. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a mangrove sediment mud-derived fungal fermentation extract with medicinal value and a preparation method and application thereof.

[0006] Specifically, as a first aspect of the present application, the present application provides a preparation method of a mangrove sediment mud-derived fungal fermentation extract, which comprises:

[0007] (1) inoculating a mangrove sediment mud-derived fungus into a liquid culture medium to obtain a spore solution; wherein the mangrove sediment mud-derived fungus is Pseudallescheria angusta SYSU-M4 is inoculated into a liquid culture medium to obtain a spore solution; wherein the mangrove sediment mud-derived fungus is Pseudallescheria angusta SYSU-M4 was deposited in the Guangdong Microbial Culture Collection Center on May 7, 2025, with the deposit number GDMCC No: 66263, and the address of the Guangdong Microbial Culture Collection Center is Building 59, No. 5, 100, Martyrs' Road, Guangzhou;

[0008] (2) inoculating the spore solution to solid culture medium, and fermenting to obtain a fermentation product;

[0009] (3) extracting the fermentation product by an extraction solvent, extracting by an extraction solvent, and separating by a separation solvent, and collecting a component with a thin layer chromatography detection relative migration value of 0.4.

[0010] The fermentation extract obtained based on the preparation method has obvious acetylcholinesterase and NO inhibitory activity, and has good anti-inflammatory effect. The preparation method has good application prospect in the preparation of anti-inflammatory drugs and anti-inflammatory health products. Moreover, the preparation method is simple in operation and suitable for large-scale production.

[0011] Specifically, in step (1), the liquid culture medium can be PDB culture medium, SBM culture medium, Martin culture medium, etc., but is not limited thereto. Preferably, in some embodiments, the liquid culture medium is seawater potato culture medium, which specifically comprises potato juice 100-800 mL / L, glucose 10-50 g / L, and sea salt 5-50 g / L.

[0012] The preparation method of the potato juice is as follows: the potato is peeled, washed, cut into pieces, and then added with deionized water and heated and boiled for 10-30 min, and then filtered to obtain the potato juice, wherein 100 g of potato is boiled to obtain 100 mL of potato juice.

[0013] Specifically, the 100-800 mL / L of potato juice in the liquid culture medium refers to that the volume of 1 L of the liquid culture medium contains 100-800 mL of potato juice, and when the volume of the liquid culture medium is less than 1 L, water is added to make up to 1 L. In addition, the unit of the amount of potato juice used in the present application is not limited to mL / L, and can be an equivalent multiple or a smaller unit. For example, it can be L / m 3 , μL / mL, but is not limited thereto.

[0014] Preferably, the composition of 1 L of the liquid culture medium is as follows: potato juice 500 mL / L, glucose 10 g / L, sea salt 10 g / L, and the balance is water.

[0015] Specifically, in step (1), the inoculation amount is 10-20 g of colony plate per 500 mL of liquid culture medium. After inoculation, it is cultured at 20-30°C for 3-4 days. Preferably, the culture temperature is 20-30°C, and the culture time is 3-4 days.

[0016] Preferably, in some embodiments, before the inoculation of the strain, a step of activating the strain is further included, which is specifically as follows: the mangrove sediment-derived fungus SYSU-M4 is placed in an incubator with a temperature of 20-30°C and a humidity of 60-90% (RH) for activation. Pseudallescheria angusta

[0017] ​Specifically, in step (2), the solid culture medium can be beef peptone medium, rice medium, potato glucose medium, etc., but is not limited thereto. Preferably, in some embodiments, the solid culture medium is rice medium, which specifically comprises: rice 20-100 g / L, seawater 40-80 mL / L, wherein the concentration of sea salt in the seawater is 20-40 g / L. It should be noted that 20-100 g / L of rice here means that 20-100 g / L of rice is placed in a 1 L fermentation device (such as a fermentation bottle), and the seawater is similar. In addition, the unit of rice usage in the present application is not limited to g / L, but can be a unit that is multiplied or reduced by an equal multiple. For example, it can be kg / m 3 , mg / mL, but is not limited thereto.

[0018] Preferably, in some embodiments, the solid culture medium comprises rice 50 g / L, seawater 50 mL / L, wherein the concentration of sea salt in the seawater is 30 g / L.

[0019] Specifically, in step (2), the inoculation amount of the spore solution is 1-8 vol%, the fermentation temperature is 20-35°C, and the fermentation time is 10-50 days. Preferably, the inoculation amount of the spore solution is 4 vol%, the fermentation temperature is 25-35°C, and the fermentation time is 25-35 days.

[0020] Specifically, in step (3), the extraction solvent is selected from methanol and / or dichloromethane, but is not limited thereto;

[0021] The extraction solvent is selected from ethyl acetate, but is not limited thereto;

[0022] The separation solvent is selected from one or more of petroleum ether-ethyl acetate, petroleum ether-isopropyl alcohol, dichloromethane-ethyl acetate, dichloromethane-ethanol, and dichloromethane-methanol, but is not limited thereto. The polarity gradient of the separation solvent increases.

[0023] Preferably, in some embodiments, the extraction solvent is selected from methanol;

[0024] The extraction solvent is selected from ethyl acetate;

[0025] The separation solvent is sequentially petroleum ether-ethyl acetate with a volume ratio of 10:0, 8:2, 7:3, 6:4, 5:5, and 4:6, ethyl acetate-methanol with a volume ratio of 10:0, 1:1, and 0:10, and dichloromethane-methanol with a volume ratio of 1:1.

[0026] Preferably, in some embodiments, step (3) comprises:

[0027] (3.1) The fermentation product is extracted 1-3 times with an extraction solvent, and the combined extract is dried to obtain a first crude extract; wherein the extraction solvent is selected from methanol;

[0028] (3.2) dissolving the first crude extract with water, and then extracting with ethyl acetate for 5-6 times, drying the combined extract to obtain a second crude extract;

[0029] (3.3) eluting the second crude extract with a normal phase silica gel column, collecting the components with a thin layer chromatography detection of a relative migration value of 0.2 to obtain an intermediate product; wherein the silica gel filler of the normal phase silica gel column is 100-200 mesh, and the eluent is petroleum ether-ethyl acetate with a volume ratio of 10:0, 8:2, 7:3, 6:4, 5:5, 4:6, ethyl acetate-methanol with a volume ratio of 10:0, 1:1, 0:10.

[0030] Specifically, through normal phase silica gel column chromatography, 36 sub-components (Fr.1-36) were obtained, and thin layer chromatography (TLC, developing agent was petroleum ether: ethyl acetate = 7:3, thin layer silica gel plate, color developing agent was vanillin) was used to analyze and combine the 36 sub-components, and the relative migration values (R f values) of each component were measured as follows: the R f value of Fr.A (sub-component 1-10) was 0.9, the R f value of Fr.B (sub-component 11-16) was 0.8, the R f value of Fr.C (sub-component 17-20) was 0.65, the R f value of Fr.D (sub-component 21-24) was 0.4, the R f value of Fr.E (sub-component 25-29) was 0.2. The main component Fr.E with a R f value of 0.2 was used as the intermediate product.

[0031] (3.4) eluting the intermediate product with a gel column, collecting the components with a thin layer chromatography detection of a relative migration value of 0.4 to obtain a fermentation extract finished product; wherein the matrix of the gel column is agarose gel with a pore size of 25-100 μm, and the eluent is dichloromethane-methanol with a volume ratio of 1:1.

[0032] Specifically, through gel column chromatography, 3 components (Fr.E-A~Fr.E-C) were obtained, thin layer chromatography (TLC, developing agent was petroleum ether: ethyl acetate = 1:1, thin layer silica gel plate, color developing agent was vanillin) was used to analyze the sub-components, and the component Fr.E-A with a relative migration value (R f value) of 0.4 was used as the finished product.

[0033] Correspondingly, as a second aspect of the present application, the present application also provides a mangrove sediment-derived fungal fermentation extract, which is prepared by the above-mentioned method for preparing a mangrove sediment-derived fungal fermentation extract. The fermentation extract has obvious acetylcholinesterase and NO inhibitory activities, and has good anti-inflammatory effects. It has good application prospects in the preparation of anti-inflammatory drugs and anti-inflammatory health products.

[0034] Based on this, as a third aspect of the present application, the present application provides the following applications of the mangrove sediment-derived fungal fermentation extract:

[0035] (1) application as an active ingredient in the preparation of a drug or a health product for inhibiting acetylcholinesterase;

[0036] (2) application as an active ingredient in the preparation of a drug or a health product for inhibiting the generation of intracellular NO;

[0037] (3) application as an active ingredient in the preparation of an anti-inflammatory drug or an anti-inflammatory health product.

[0038] The implementation of the present application has the following beneficial effects:

[0039] The present application uses a mangrove sediment-derived fungus Pseudallescheria angusta SYSU-M4 is cultured in a liquid culture medium, and fermented in a solid culture medium. The fermentation product is extracted by an extraction solvent, extracted by an extraction solvent, and separated by a separation solvent. A component with a thin layer chromatography detection Rf value of 0.4 is obtained, i.e. a fermentation extract. The fermentation extract has obvious acetylcholinesterase and NO inhibitory activities, and has good anti-inflammatory effects. It has good application prospects in the preparation of anti-inflammatory drugs and anti-inflammatory health products. Moreover, the preparation method of the present application is simple in operation and suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is an HPLC chart of the mangrove sediment-derived fungal fermentation extract in Example 1 of the present application;

[0041] Figure 2 is a cytotoxicity experiment result chart of the mangrove sediment-derived fungal fermentation extract in Example 3 of the present application;

[0042] Figure 3 is a chart of the inhibitory effect of the mangrove sediment-derived fungal fermentation extract on LPS-induced intracellular NO of BV2 cells in Example 4 of the present application. DETAILED DESCRIPTION

[0043] In order to make the object, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0044] Example 1 Mangrove sediment-derived fungal fermentation extract and method of preparation thereof

[0045] Mangrove sediment-derived fungus in this example Pseudallescheria angusta SYSU-M4 was deposited at the Guangdong Microbial Culture Collection Center on May 7, 2025, with the deposit number GDMCC No: 66263. The address of the Guangdong Microbial Culture Collection Center is: 59 Building, 5th Floor, 100, Martyrs' Road, Guangzhou, Guangdong Province.

[0046] The specific preparation method is as follows:

[0047] (1) The mangrove sediment-derived fungus SYSU-M4 was activated overnight in a mold culture box at a temperature of 28°C and a humidity of 80%. Pseudallescheria angusta The SYSU-M4 cryovial was placed in a mold culture box at a temperature of 28°C and a humidity of 80% for activation overnight.

[0048] (2) The activated strain was inoculated into pre-sterilized seawater potato culture medium (potato juice 500 mL, glucose 10 g, sea salt 30 g, add pure water to 1 L, 1x10 5 Pa sterilization for 30 min) and cultured on a shaking table (100 rpm) for 4 days to obtain a spore solution.

[0049] (3) The spore solution was inoculated into a conical flask (1 L) containing rice culture medium (rice 50 g / flask, sea salt content 30 g / L of seawater 50 mL / flask, 121°C, 1x10 5 Pa sterilization for 30 min) and cultured at room temperature for 30 days until the mycelium completely filled the culture medium and fermentation was completed.

[0050] (4) The fermented cake was treated with methanol for extraction three times, and the combined extract was evaporated to dryness to obtain a first crude extract.

[0051] (5) The first crude extract was redissolved in deionized water, and extracted with ethyl acetate as the extractant for 5-6 times. The combined extract was concentrated and evaporated to dryness to obtain a second crude extract.

[0052] (6) The second crude extract was subjected to column chromatography using normal phase silica gel. Specifically, the second crude extract was thoroughly mixed with blank silica gel (100-200 mesh) at a weight ratio of 1:2 to obtain sample silica gel, which was packed into a column using a wet method. First, 100-200 mesh blank silica gel was added, and then the sample silica gel was laid flat on top (sample silica gel:blank silica gel at a volume ratio of 1:7). A layer of cotton was laid on top of the column to prevent the flat surface from being washed away by the solvent. The column was then eluted with petroleum ether-ethyl acetate (volume ratio of 10:0, 8:2, 7:3, 6:4, 5:5, and 4:6), and ethyl acetate-methanol (10:0, 1:1, and 10:0) in gradient. A total of 36 sub-fractions (Fr. 1-36) were obtained. The characteristics of each fraction were analyzed by thin layer chromatography (TLC, developing agent: petroleum ether: ethyl acetate = 7:3, thin layer silica gel plate, color developing agent: vanillin). Based on the similarity of polarity, the fractions were merged into five main fractions, Fr. A-Fr. E. The main fraction Fr. E with a retardation value (R f value) of 0.2 was used as an intermediate product.

[0053] (7) The intermediate product was eluted using a gel column to obtain the final fermented extract. Specifically, the gel column was Sephadex LH-20, and the eluent was dichloromethane-methanol at a volume ratio of 1:1. Through gel column chromatography, a total of 3 fractions (Fr. E-A-Fr. E-C) were obtained. The characteristics of each fraction were analyzed by thin layer chromatography (TLC, developing agent: petroleum ether: ethyl acetate = 1:1, thin layer silica gel plate, color developing agent: vanillin). The fraction Fr. E-A with a retardation value (R f value) of 0.4 was used as the final product. The HPLC of the final fermented extract obtained in this example is shown in Table 1. Figure 1

[0054] Example 2 Inhibition of acetylcholinesterase by fermented extract from mangrove sediment-derived fungi

[0055] 1. Experimental method

[0056] Primary screening: 5 mg / mL of the fermented extract was dissolved in methanol and added to a 96-well plate. After the organic solvent was completely volatilized, DMSO, PBS, AChE, and DTNB were added to each reaction well in turn. After incubation at 37°C for 10 minutes, ATCI was added for further incubation for 20 minutes. The absorbance at 405 nm was measured and recorded as the test group. At the same time, a blank control group without AChE and an experimental control group containing only DMSO, PBS, AChE, and DTNB were set up, as well as a blank group without AChE. After processing the experimental data based on the inhibition rate calculation formula, 50% was set as the screening threshold, and samples meeting the standard were retained for the secondary screening experiment.

[0057] Secondary screening: half-log dilution method. The concentration-log curve was fitted using Origin 9.1 software, and the ln(IC​50 IC of AChE obtained 50 values in Origin 9.1 software by fitting the concentration logarithmic curve and calculating ln(IC 50 ), finally all the data were analyzed and calculated for IC 50 values in Excel.

[0058]

[0059] wherein, A 空白 is the absorbance of the blank group, A 空白对照 is the absorbance of the blank control group, A 实验 is the absorbance of the experimental group, A 实验对照 is the absorbance of the experimental control group.

[0060] 2、Experimental results

[0061] The specific experimental results are shown in the following table.

[0062]

[0063] Note: a is the positive control

[0064] As can be seen from the above table, the fermentation extract prepared in Example 1 showed acetylcholinesterase inhibitory activity, with an IC 50 value of 83.4 μ mol / L (positive control: Huperzine A IC 50 value of 22 μ mol / L).

[0065] Example 3 Cytotoxicity experiment of fermentation extract of fungus from mangrove sediment

[0066] 1、Experimental method:

[0067] The BV2 cell line was obtained from the Chinese Academy of Sciences Cell Bank (China, Shanghai), and the culture medium was DMEM medium (Gibco, item number: 11995065) 90 mL, with the addition of fetal bovine serum FBS (Gibco) 10 mL, and was cultured in a 37°C CO2concentration of 5% incubator. The potential toxicity of the compound represented by formula (I) on cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl bromide tetrazolium (MTT) method.

[0068] The preparation method of BV-2 cell complete culture medium is: adding 10 mL of fetal bovine serum (FBS) and 1 mL of 100× penicillin-streptomycin double-antibiotic solution to every 100 mL of high-sugar DMEM medium, thoroughly mixing, and then passing through a 0.22 μM filter membrane filtration sterilization, 4℃ preservation for standby. This medium formula can provide the nutrients required for cell growth, while effectively preventing bacterial contamination.

[0069] Cell inoculation: Cells were inoculated in 96-well plates at a density of 1 x 104cells per well, ensuring uniform distribution and adhesion growth of cells.

[0070] Drug treatment: After cell inoculation, the test sample was added at a final concentration of 1-20 μ g / mL. Multiple replicate wells were set up for each concentration to improve the reliability of the experimental results. Colorimetric detection: After 24 hours of drug treatment, MTT reagent was added to each well and incubated for another 4 hours. Subsequently, the culture medium was removed and the formed formazan crystals were dissolved using DMSO. Finally, the absorbance (OD value) of each well was measured at a wavelength of 490 nm.

[0071] Result calculation: The percentage of cell viability was calculated according to the following formula: Cell viability (%) = (experimental group OD value / control group OD value) x 100%. By comparing the OD values of the experimental and control groups, the effect of different concentrations of samples on cell viability was evaluated to determine their potential cytotoxicity.

[0072] 2. Experimental results:

[0073] The fermentation extract obtained in Example 1 was used to treat BV2 cells at doses of 1, 10, and 20 μ g / mL. The cell viability of each dose group was similar to that of the 0 μ g / mL control group, with no significant difference (P>0.05). Figure 2

[0074] Example 4: Inhibition of NO by Mangrove Sediment-derived Fungal Fermentation Extract

[0075] BV2 cell line, medium information as in Example 2.

[0076] Standard curve preparation: According to the instructions of the kit (Beyotime S0021M), a standard curve was prepared. The established standard curve regression equation y=0.0036x+0.0542 had a correlation coefficient R2=0.999, indicating a good linear relationship.

[0077] Experimental grouping: The experimental design included three main groups: normal control group, LPS stimulation group (concentration of 1 μ g / mL), and sample treatment group (concentration range from 1 to 20 μ g / mL). Five replicate wells were set up for each group to ensure the reliability of the data.

[0078] ​​​Detection step: Firstly, collect the cell supernatant of each experimental group. Then, mix equal volume of cell supernatant with Griess reagent, and react for 10 minutes under the condition of avoiding light. Finally, measure the absorbance value at wavelength 540 nm using microplate reader.

[0079] Statistical analysis: The obtained data was statistically analyzed by One-way ANOVA. The significant level compared with LPS stimulation group was marked as *p<0.05, **p<0.01, ***p<0.001, respectively, to represent the statistical significance of NO content difference between different treatment groups.

[0080] 2. Experimental results:

[0081] The average concentration of NO of the fermentation extract prepared in Example 1 was 23.0, 20.3, 18.9 μ M after treatment at the dose of 1, 10 and 20 μ g / mL, respectively. Figure 3 The average concentration of NO of the blank group (0 μ g / mL) and the control group (LPS) was 23.07, 20.73 μ M, respectively. This shows that the fermentation extract can significantly inhibit the generation of NO at the concentration of 20 μ g / mL.

[0082] The above is the preferred embodiment of the application. It should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements are also considered within the protection scope of the application.

Claims

1. A method for preparing a fungal fermentation extract derived from mangrove sediment mud, characterized in that, include: (1) Fungi derived from mangrove sediment ( Pseudallescheria angusta SYSU-M4 was inoculated into liquid culture medium to obtain spore solution; wherein, the fungus derived from the mangrove sediment mud ( Pseudallescheria angusta SYSU-M4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 7, 2025, with accession number GDMCC No: 66263. The address of the Guangdong Provincial Center for Microbial Culture Collection is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. (2) The spore liquid is inoculated into a solid culture medium and fermented to obtain the fermentation product; (3) The fermentation product is extracted with an extraction solvent, extracted with an extraction solvent, and separated by separation solvent chromatography. The fraction with a specific shift value of 0.4 is collected by thin-layer chromatography. The extraction solvent is methanol; The extraction solvent is ethyl acetate; The separation solvents are, in order, petroleum ether-ethyl acetate with volume ratios of 10:0, 8:2, 7:3, 6:4, 5:5, and 4:6, ethyl acetate-methanol with volume ratios of 10:0, 1:1, and 0:10, and dichloromethane-methanol with a volume ratio of 1:

1.

2. The method for preparing the fungal fermentation extract derived from mangrove sediment mud as described in claim 1, characterized in that, The liquid culture medium comprises: 100-800 mL / L potato juice, 10-50 g / L glucose, and 5-50 g / L sea salt; The solid culture medium comprises 20-100 g / L rice and 40-80 mL / L seawater, wherein the concentration of sea salt in the seawater is 20-40 g / L; In step (1), the culture temperature is 20~30℃ and the culture time is 3~4 days; In step (2), the fermentation temperature is 20~35℃ and the fermentation time is 10~50 days.

3. The method for preparing the fungal fermentation extract derived from mangrove sediment mud as described in claim 1, characterized in that, The liquid culture medium comprises: 500 mL / L potato juice, 10 g / L glucose, and 10 g / L sea salt; The solid culture medium consists of 50 g / L rice and 50 mL / L seawater, wherein the concentration of sea salt in the seawater is 30 g / L. In step (1), the culture temperature is 20~30℃ and the culture time is 3~4 days; In step (2), the fermentation temperature is 25~35℃ and the fermentation time is 25~35 days.

4. The method for preparing the fungal fermentation extract derived from mangrove sediment as described in claim 1, characterized in that, Step (3) includes: (3.1) Extract the fermentation product 1 to 3 times with an extraction solvent, combine the extracts and dry them to obtain the first crude extract; wherein, the extraction solvent is methanol; (3.2) Dissolve the first crude extract in water, then extract it 5 to 6 times with ethyl acetate, combine the extracts and dry them to obtain the second crude extract; (3.3) The second crude extract was eluted using a normal-phase silica gel column, and the fractions with a TLC detection ratio of 0.2 were collected to obtain the intermediate product; wherein the silica gel packing material used in the normal-phase silica gel column was 100~200 mesh, and the eluents used were petroleum ether-ethyl acetate with volume ratios of 10:0, 8:2, 7:3, 6:4, 5:5, and 4:6, and ethyl acetate-methanol with volume ratios of 10:0, 1:1, and 0:10, respectively; (3.4) The intermediate product is eluted using a gel column, and the fraction with a specific shift value of 0.4 is collected by thin-layer chromatography to obtain the fermentation extract product; wherein the matrix of the gel column is agarose gel with a pore size of 25~100μm; the eluent used is dichloromethane-methanol with a volume ratio of 1:

1.

5. A fungal fermentation extract derived from mangrove sediment mud, characterized in that, It is prepared by the method for preparing fungal fermentation extract derived from mangrove sediment mud as described in any one of claims 1 to 4.

6. Application of fungal fermentation extracts derived from mangrove sediment as active ingredients in the preparation of anti-inflammatory drugs; in, The mangrove sediment-derived fungal fermentation extract is prepared by the method for preparing the mangrove sediment-derived fungal fermentation extract as described in any one of claims 1 to 4; Fungi originating from mangrove sediments ( Pseudallescheria angusta SYSU-M4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 7, 2025, with accession number GDMCC No: 66263. The address of the Guangdong Provincial Center for Microbial Culture Collection is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

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