Two sesquiterpenoid compounds derived from mangrove fungi, their preparation methods, and their applications in anti-inflammatory drugs

Abstract

The present invention relates to the technical field of anti-inflammatory drugs, and in particular to two sesquiterpenoid compounds derived from mangrove fungi, a preparation method thereof, and an application thereof in anti-inflammatory drugs. The structural formulas of the two sesquiterpenoid compounds are shown in formula (I) and formula (II). The method comprises the following steps: inoculating the mangrove fungi strain Aspergillus sp. GXNU-G12 into a liquid culture medium, culturing on a shaking table, and obtaining a seed culture solution; inoculating the strain with good growth status in the seed culture solution into a rice fermentation culture medium, culturing statically, and obtaining fungal mycelium; extracting the fungal mycelium three times with methanol, extracting with ethyl acetate, and concentrating the extract to obtain an extract; separating the extract by using a column chromatography separation technology, and recrystallizing to obtain the sesquiterpenoid compounds.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-inflammatory drugs, in particular to two sesquiterpenoid compounds derived from mangrove fungi, a preparation method thereof and application thereof in anti-inflammatory drugs. Background Art

[0002] The ocean's unique environmental conditions of high temperature, high salinity, and high pressure give organisms living in this special environment specialized metabolic pathways, enabling them to produce a large number of novel and highly active compounds, making them an important source of drug lead compounds. To date, a large number of new secondary metabolites with anti-inflammatory activity have been widely reported from marine fungi. Marine natural products have also attracted increasing attention due to their unique anti-inflammatory mechanisms and are considered hot spots for anti-inflammatory drug development. Many marine natural products with anti-inflammatory activity have been developed into anti-inflammatory drugs for clinical use. In the mid-1980s, manoalide, isolated from marine natural products, was successfully developed into a phosphatase A2 inhibitor and is currently in clinical trials as a typical anti-inflammatory agent. Cephalosporin C is used clinically as an anti-inflammatory drug to treat otitis media, tonsillitis, acute and chronic bronchitis, and other inflammatory conditions. The marine anti-inflammatory drug coltitrole is used to treat asthma and inflammation, and its derivative, the steroid IPL576092, has also entered Phase II clinical trials as a new anti-inflammatory drug. The eye drop Carragelos is clinically used to treat viral conjunctivitis, among other conditions.

[0003] Inflammation is the body's defensive response to stimuli, underlying various physiological and pathological processes and a key factor in complex diseases and disorders, including autoimmune diseases, metabolic syndrome, neurodegenerative diseases, cancer, and cardiovascular disease. With the accelerating pace of urbanization, aging, and changing lifestyles, inflammatory diseases have become a major threat to global health. These diseases include many infectious diseases, as well as major chronic non-communicable conditions such as autoimmune diseases, malignant tumors, and cardiovascular disease. It is estimated that approximately 2 billion people worldwide suffer from various forms of inflammatory diseases. Anti-inflammatory drugs are an important treatment for inflammatory diseases, primarily categorized as nonsteroidal anti-inflammatory drugs (NSAIDs) and nonsteroidal anti-inflammatory drugs (NSAIDs). The development of the pharmaceutical industry is driving continuous innovation in the research and development and application of anti-inflammatory drugs worldwide. With the continuous improvement of medical standards and technological advancements, the anti-inflammatory drug market holds a promising future. The current state of the anti-inflammatory drug industry indicates that the total market size exceeds US$320 billion, demonstrating its significant market presence. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, and acetaminophen, are the most widely used anti-inflammatory drugs in clinical practice. However, long-term use can produce a range of adverse reactions, including gastrointestinal and skin irritation, changes in blood composition, increased burden on the liver and kidneys, and cardiovascular disease. There is an urgent need to discover anti-inflammatory drugs with better efficacy and fewer side effects, which are essential for the treatment of inflammatory diseases. Summary of the Invention

[0004] The object of the present invention is to address the above-mentioned problems and provide two sesquiterpenoid compounds derived from mangrove fungi, their preparation methods and their use in anti-inflammatory drugs.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] Two sesquiterpenoid compounds derived from mangrove fungi, the structural formulas of the two sesquiterpenoid compounds are shown in formula (I) and formula (II):

[0007]

[0008] The two sesquiterpenoid compounds (I) and (II) provided by the present invention are isolated and obtained from the solid fermentation product of the mangrove fungus strain Aspergillus sp. GXNU-G12. The mangrove fungus strain Aspergillus sp. GXNU-G12 was deposited in the Guangdong Provincial Microbiological Culture Collection on June 16, 2024, with a deposit number of GDMCC No: 60801.

[0009] Specifically, the preparation method of the two sesquiterpenoid compounds derived from mangrove fungi comprises the following steps:

[0010] (1) Seed culture: inoculating the mangrove fungus strain Aspergillus sp. GXNU-G12 into a liquid culture medium and culturing on a shaking table to obtain a seed culture solution;

[0011] (2) Fermentation culture: The strain with good growth status in the seed culture solution is transferred into the rice fermentation medium and cultured statically to obtain fungal mycelium;

[0012] (3) Extraction: The fungal mycelium obtained in step (2) was extracted three times with methanol, extracted with ethyl acetate, and the extract was concentrated to obtain an extract;

[0013] (4) Separation and recrystallization: The extract is separated by column chromatography, and gradient elution is performed using petroleum ether-ethyl acetate as the eluent; the fraction collected from 30% petroleum ether-ethyl acetate is recrystallized with methanol to obtain a sesquiterpenoid compound as shown in formula (I); the fraction collected from 40% petroleum ether-ethyl acetate is recrystallized with methanol-ethyl acetate to obtain a sesquiterpenoid compound as shown in formula (II).

[0014] In the above preparation method, preferably, the liquid culture medium in step (1) comprises 20-40 g of glucose, 30-50 g of potatoes, and 1-2 liters of water.

[0015] In the above preparation method, preferably, the shaking culture conditions in step (1) are a shaking speed of 80-140 rpm and a culture temperature of 25-30° C. for 3-7 days.

[0016] In the above preparation method, preferably, the rice culture medium used for the fermentation culture in step (2) is obtained by mixing rice and seawater in a mass volume ratio of 50g:50ml.

[0017] In the above preparation method, preferably, the conditions for the static culture in step (2) are to control the temperature to 25°C-30°C and the culture time to 28-40 days.

[0018] In the above preparation method, preferably, the column chromatography separation technology in step (3) is a normal phase silica gel column chromatography technology, wherein the normal phase silica gel column chromatography uses petroleum ether and ethyl acetate as eluents, and the ratios are 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60 respectively; and the recrystallization is a room temperature dissolution and crystallization technology.

[0019] The sesquiterpenoid structures (I) and (II) derived from the mangrove fungus have anti-inflammatory activity and can be used in the preparation of anti-inflammatory drugs.

[0020] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0021] 1. The present invention is the first to use the mangrove fungus Aspergillus sp. GXNU-G12 to obtain two sesquiterpenoid compounds of formula (I) and formula (II). These compounds have significant NO inhibitory activity. NO (nitric oxide) inhibitory activity screening test showed that compounds (I) and (II) showed significant inhibitory activity on NO production, and the half inhibitory concentration (IC50) of compound (I) was 50 The half-maximal inhibitory concentration (IC 50 value) was 8.20 μM, which was better than the positive control indomethacin (IC 50 =24.88 μM). Therefore, the two sesquiterpenoid compounds (I) and (II) provided by the present invention can be considered as potential lead compounds for the development of anti-inflammatory drugs and have clinical application potential for anti-inflammatory treatment.

[0022] 2. The present invention utilizes mangrove fungus Aspergillus sp. GXNU-G12 to prepare sesquiterpenoid compounds, which has a simple preparation process, a short production cycle, low cost, no pollution to the environment, and is easy to mass-produce. DETAILED DESCRIPTION

[0023] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0024] 1. Preparation Example

[0025] Example 1

[0026] Isolation and Identification of Mangrove Fungus Aspergillus sp. GXNU-G12

[0027] Materials: Leaves of the mangrove plant Acanthus tatarinowii collected from Beihai City, Guangxi.

[0028] After culturing, isolating, and identifying the strain, a pure fungal strain was obtained and identified as Aspergillus sp. GXNU-G12. The mangrove fungus Aspergillus sp. GXNU-G12 was deposited with the Guangdong Provincial Microbiological Culture Collection on June 16, 2024, with the deposit number GDMCC No. 60801. The depository's address is 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0029] Example 2

[0030] The preparation method of two sesquiterpenoid compounds comprises the following steps:

[0031] (1) Seed culture: The medium used for seed culture was composed of 20 g glucose, 30 g potato, and 1 liter water. Aspergillus sp. GXNU-G12 was selected and inoculated into a culture flask. The culture was shaken at 80 rpm and 25°C for 3 days to obtain a seed culture solution.

[0032] (2) Fermentation culture: The strain in the seed culture solution was transferred into a solid rice fermentation medium, wherein the medium was a mixture of rice and seawater in a mass volume ratio of 50 g:50 ml, and the culture was allowed to stand at a temperature of 25°C-30°C for 28 days to obtain fungal mycelium;

[0033] (3) Extraction: After fermentation, the fungal mycelium obtained in step (2) was extracted three times with methanol, the extract was suspended in water, and then extracted with ethyl acetate, and the extract was concentrated to obtain an extract (20 g);

[0034] (4) Separation and recrystallization: The extract was first separated by normal phase silica gel column chromatography, using petroleum ether-ethyl acetate as eluent, and gradient elution was performed in the ratio of 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60 in sequence; the fraction collected from 30% petroleum ether-ethyl acetate was recrystallized with methanol to obtain a sesquiterpenoid compound as shown in formula (I); the fraction collected from 40% petroleum ether-ethyl acetate was recrystallized with methanol-ethyl acetate to obtain a sesquiterpenoid compound as shown in formula (II).

[0035] Example 3

[0036] The preparation method of two sesquiterpenoid compounds comprises the following steps:

[0037] (1) Seed culture: The medium used for seed culture was composed of 40 g of glucose, 50 g of potato, and 2 liters of water. Aspergillus sp. GXNU-G12 was selected and inoculated into a culture flask. The culture was shaken at 140 rpm and 30°C for 7 days to obtain a seed culture solution.

[0038] (2) Fermentation culture: The strain in the seed culture solution was transferred into a solid rice fermentation medium, wherein the medium was a mixture of rice and seawater in a mass volume ratio of 50 g:50 ml, and the culture was allowed to stand at a temperature of 25°C-30°C for 40 days to obtain fungal mycelium;

[0039] (3) Extraction: After fermentation is completed, the fungal mycelium obtained in step (2) is extracted three times with methanol, the extract is suspended in water, and then extracted with ethyl acetate, and the extract is concentrated to obtain an extract;

[0040] (4) Separation and recrystallization: The extract was first separated by normal phase silica gel column chromatography, using petroleum ether-ethyl acetate as eluent, and gradient elution was performed in the ratio of 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60 in sequence; the fraction collected from 30% petroleum ether-ethyl acetate was recrystallized with methanol to obtain a sesquiterpenoid compound as shown in formula (I); the fraction collected from 40% petroleum ether-ethyl acetate was recrystallized with methanol-ethyl acetate to obtain a sesquiterpenoid compound as shown in formula (II).

[0041] 2. Confirmation of Compounds

[0042] The sesquiterpenoid compounds obtained above were dissolved in deuterated chloroform and deuterated methanol, and identified by nuclear magnetic resonance spectroscopy (NMR) and other methods to obtain the following test data:

[0043] Sesquiterpenoid I: colorless powder, soluble in chloroform. HRESIMS: m / z 279.1228 [M+H] + (calcdfor279.1232C 15 H 19 O5 + ). 1 H NMR (400 MHz, CDCl3) δ H 6.66(1H,s,H-11),4.84(1H,dt,J=16.4,2.6Hz,H-13α),4.76(2H,dd,J=16.4,2.6Hz,H-13β), 5.78(1H,dd,J=11.2,2.7Hz,H-8),2.62(1H,m,H-3α),2.45(1H,m,H-3β),6.27(1H,m,H-7),2. 68(1H,ddd,J=16.2,6.7,2.7,H-6α),2.44(1H,ddd,J=16.2,6.7,2.7,H-6β),1.71(1H,td,J=1 4.0,4.0Hz,H-4α),1.64(1H,dt,J=14.0,4.0Hz,H-4β),4.40(2H,s,H-15),4.17(2H,s,H-14); 13 C NMR (100 MHz, CDCl3) δ C177.20(C-1),167.41(C-10),151.70(C-12),128.53(C-2),117.0(C-11),76.70(C-9),134.23(C-8),73. 11(C-13),44.10(C-5),35.95(C-4),39.51(C-6),62.04(C-15),63.90(C-14),132.73(C-7),23.6(C-3).

[0044] Sesquiterpenoid II: colorless powder, soluble in chloroform. HRESIMS: m / z 261.1128 [M+H] + (calcdfor261.1127C 15 H 17 O4 + ). 1 H NMR (400 MHz, CDCl3) δ H 6.68(1H,s,H-11),4.81(1H,dt,J=16.4,2.6Hz,H-13α),4.72(2H,dd,J=16.4,2.6Hz,H-13β), 5.88(1H,dd,J=11.2,2.7Hz,H-8),2.44(1H,m,H-3α),2.49(1H,m,H-3β),6.17(1H,m,H-7),2. 77(1H,ddd,J=16.2,6.7,2.7,H-6α),2.60(1H,ddd,J=16.2,6.7,2.7,H-6β),1.78(1H,td,J=1 4.0,4.0Hz,H-4α),1.69(1H,dt,J=14.0,4.0Hz,H-4β),4.50(2H,s,H-15),4.35(2H,s,H-14); 13 C NMR (100 MHz, CDCl3) δ C 177.33(C-1),167.40(C-10),151.51(C-12),128.27(C-2),115.6(C-11),76.2(C-9),133.00(C-8),73. 11(C-13),44.10(C-5),35.95(C-4),39.51(C-6),65.11(C-15),64.50(C-14),133.40(C-7),23.4(C-3).

[0045] After identification, the structural formulas of the two sesquiterpenoid compounds are shown in formula (I) and (II):

[0046]

[0047] 3. Anti-inflammatory activity experiment of two sesquiterpenoid compounds (inhibitory effect on cell NO production)

[0048] Experiment on the inhibitory effect of the two sesquiterpenoid compounds obtained in Examples 2 and 3 on NO production in RAW264.7 cells:

[0049] 1. Materials:

[0050] 1.1DMEM high-glucose medium, fetal bovine serum, trypsin (Gibco, USA); phosphate-buffered saline (PBS) (Gibco, USA); dimethyl sulfoxide (MPBio, USA); CCK8 cell counting kit (APExBIO, USA); dexamethasone and lipopolysaccharide (LPS) (Sigma, USA); Griess kit (Beyond Biotechnology, Shanghai, China).

[0051] 1.2 MTT assay to determine the toxicity of the two compounds on RAW264.7 cells:

[0052] The compound was dissolved in dimethyl sulfoxide (100%) to a concentration of 20 mmol·L -1 Store in the refrigerator for later use. Dilute to 200 μmol·L with DMEM high glucose culture medium during the experiment. -1 RAW264.7 cells were digested with 0.25% trypsin and resuspended in DMEM high glucose medium containing 10% FBS at a concentration of 1.25×10 4 Cells (100 μL per well) were seeded into 96-well plates and cultured in a 37°C constant temperature incubator with 5% CO2 for 24 hours. The culture medium of each group was replaced with fresh DMEM high glucose culture medium containing 10% FBS. The compound test group was added with the corresponding compound (10 μL per well), and the normal control group was added with DMEM high glucose culture medium containing 10% fetal bovine serum (10 μL per well) and cultured for another 24 hours. 5 mg·mL -1 MTT (10 μL per well) was added for live cell staining, with resveratrol used as a control. After 3 hours of incubation, the culture medium was discarded and DMSO (100 μL per well) was added to dissolve the cells. The cells were shaken on a plate shaker to fully dissolve the cells. The OD value was then measured at a wavelength of 490 nm. For the blank control group, 100 μL of DMSO was added to each well. The cell viability of each group was calculated using the following formula:

[0053] Cell survival rate (%) = (OD value 化合物测试组 -OD value 空白组 ) / (OD value 正常对照组 -OD value 空白组 )×100%.

[0054] The experiment found that when the concentrations of the two compounds were screened at 20μM, 40μM, and 80μM, respectively, the survival rates of RAW264.7 cells were greater than or close to 100%, indicating that their toxicity to RAW264.7 cells met the requirements for conducting anti-inflammatory experiments.

[0055] 1.3 NO inhibition test

[0056] RAW264.7 cells were cultured at 2 × 10 cells per ml. 4 The cells were seeded at a density of 100 μL per well in a 96-well culture plate and cultured in a 37°C constant temperature incubator with 5% CO2 for 24 hours. The culture medium of each group was replaced with fresh DMEM high glucose culture medium containing 10% fetal bovine serum. The corresponding concentration of the test compound was added to the drug administration group (10 μL per well), and the normal control group and lipopolysaccharide model group were added with DMEM high glucose culture medium containing 10% fetal bovine serum (10 μL per well). Subsequently, 0.001 mg·mL -1 The control group was treated with 10 μL of lipopolysaccharide (10 μL per well). The normal control group was treated with 10 μL of DMEM high-glucose culture medium containing 10% FBS. After 24 hours of incubation, 50 μL of supernatant was added to each well and reacted with 50 μL of Greiss reagent at room temperature for 15 minutes. The OD value was measured at a wavelength of 540 nm. The molar mass fraction of nitric oxide (NO) in each group was calculated using a standard curve for nitric oxide (NO). The inhibition rate of each drug-treated group was further calculated using the following formula:

[0057] NO generation inhibition rate (%) = 1-w1÷w2×100%

[0058] w1 represents the NO molar mass fraction of the compound test group, and w2 represents the NO molar mass fraction of the lipopolysaccharide group.

[0059] 2. Test results

[0060] The experimental results showed that compounds (I) and (II) could significantly and effectively inhibit the production of NO in cells. The inhibitory effect of the compounds on NO production in RAW264.7 cells was IC 50 The values ​​(μM) are shown in Table 1.

[0061] Table 1 Inhibitory effect of compounds on LPS-induced NO in RAW264.7 cells

[0062]

[0063] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. Two sesquiterpenoid compounds derived from mangrove fungi, characterized in that: The structural formulas of the two sesquiterpenoid compounds are shown in formula (I) and formula (II): 。 2. A method for preparing two sesquiterpenoid compounds derived from mangrove fungi according to claim 1, characterized in that: The two sesquiterpenoid compounds (I) and (II) are isolated from the mangrove fungus strain Aspergillus sp. GXNU-G12 solid fermentation isolated, mangrove fungus strains Aspergillus sp. GXNU-G12 was deposited in Guangdong Provincial Microbiological Culture Collection on June 16, 2024, with the deposit number GDMCC No: 60801; The following steps are involved: (1) Seed culture: the mangrove fungus strain Aspergillus sp. GXNU-G12 was inoculated into liquid culture medium and cultured on a shaking platform to obtain seed culture solution; (2) Fermentation culture: The strain with good growth status in the seed culture solution is transferred into the rice fermentation medium and cultured statically to obtain fungal mycelium; (3) Extraction: extracting the fungal mycelium obtained in step (2) three times with methanol, extracting with ethyl acetate, and concentrating the extract to obtain an extract; (4) Separation and recrystallization: The extract was separated by normal phase silica gel column chromatography, using petroleum ether-ethyl acetate as eluent, and gradient elution was performed in the ratio of 0:100, 5:95, 10:90, 20:80, 30:70, and 40:60 in sequence; the fraction collected from 30% petroleum ether-ethyl acetate was recrystallized with methanol to obtain a sesquiterpenoid compound as shown in formula (I); the fraction collected from 40% petroleum ether-ethyl acetate was recrystallized with methanol-ethyl acetate to obtain a sesquiterpenoid compound as shown in formula (II).

3. The preparation method according to claim 2, wherein: The composition of the liquid culture medium in step (1) is as follows: 20-40 g of glucose, 30-50 g of potatoes, and 1-2 liters of water.

4. The preparation method according to claim 2, wherein: The shaking culture conditions in step (1) are a shaking speed of 80-140 rpm and a culture temperature of 25-30°C for 3-7 days.

5. The preparation method according to claim 2, wherein: The rice culture medium used in the fermentation culture in step (2) is obtained by mixing rice and seawater in a mass volume ratio of 50 g:50 ml.

6. The preparation method according to claim 2, wherein: The conditions for the static culture in step (2) are to control the temperature to 25°C-30°C and the culture time to 28-40 days.

7. Use of the compounds of sesquiterpenoid structures (I) and (II) derived from mangrove fungi according to claim 1 in the preparation of anti-inflammatory drugs.

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