Application of monomethylsulfonamide in the preparation of drugs for treating ulcerative colitis

Abstract

This invention relates to the field of pharmaceutical technology, specifically disclosing the application of monomethylsulochrin in the preparation of drugs for treating ulcerative colitis. Experimental studies have shown that monomethylsulochrin can significantly improve symptoms such as weight loss, diarrhea, bloody stools, and shortened colon length in DSS-induced ulcerative colitis (UC) mice, alleviate inflammatory symptoms in UC mice, effectively improve the pathological damage of colonic tissue in UC mice, effectively inhibit the expression of inflammatory factors in UC mice, and reduce the inflammatory response in UC mice. Research indicates that monomethylsulochrin has a significant and definite therapeutic effect on ulcerative colitis and can be used to prepare drugs for treating ulcerative colitis. This invention provides a new drug source for the prevention and treatment of ulcerative colitis and broadens the application field of monomethylsulochrin.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of monomethylsulfonylpyrrolidone in the preparation of drugs for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease that primarily affects the mucosa of the colon and rectum. It is characterized by inflammation and ulceration of the colonic wall, leading to symptoms such as diarrhea, abdominal pain, and rectal bleeding. The exact cause of the disease is unclear, but it may be related to genetics, immune system abnormalities, and environmental factors. With its increasing incidence, it places a significant burden on global public health. Currently, Western medicine treatments for ulcerative colitis mainly include salicylates, immunosuppressants, glucocorticoids, and biological inhibitors, but all have significant side effects and a high risk of relapse after discontinuation. For example, 5-aminosalicylic acid drugs may cause loss of appetite, heartburn, nausea, and vomiting. Long-term use of glucocorticoids may lead to osteoporosis and femoral head necrosis. Immunosuppressants and biological agents may pose risks such as hypertension, opportunistic infections, and nephrotoxicity. While relatively safe stem cell therapy is limited in clinical application due to its long treatment time and high cost, there is an urgent need to find safer, more effective, and less expensive treatments. Traditional Chinese medicine (TCM) treatment for UC is characterized by its flexibility in medication, multiple effects, fewer toxic side effects, and low cost, making it worthwhile to invest effort in exploring its effectiveness and safety.

[0003] Natural products represent a vast treasure trove for new drug development. Their diverse structural types (including steroids, polyketides, nonribosomal peptides, terpenes, and alkaloids) and significant biological activities (including antioxidant, anti-inflammatory, antitumor, and antibacterial effects) make them crucial as lead-in drugs for treating human diseases. Among these, the unique ecological environment of the ocean fosters the distinctive metabolic mechanisms of marine microorganisms, leading to the production of novel and uniquely active metabolites, which are a vital source of new marine natural products. These compounds possess significant pharmacological activities, such as antitumor and anti-inflammatory effects. Marine natural products have emerged as one of the most promising new areas in drug development.

[0004] The tung tree is an important tree species in mangrove forests, and studies have shown that it possesses anti-inflammatory pharmacological activity. The compound Monomethylsulochrin (MMSC) is derived from an endophytic fungus, *Aspergillus fumigatus*, found on mangrove tung trees. Aspergillus fumigatus The secondary metabolite of ) has the molecular formula C 18 H 18O7, or monomethylsulfonyl ... Summary of the Invention

[0005] The purpose of this invention is to provide the application of monomethylsulochrin in the preparation of drugs for treating ulcerative colitis. This invention discloses a new use of monomethylsulochrin, providing a new source of drugs with high safety and good efficacy for the prevention and treatment of ulcerative colitis.

[0006] The technical solution adopted in this invention is as follows:

[0007] The application of monomethylsulochrin in the preparation of drugs for treating ulcerative colitis. The English name of the monomethylsulochrin is: Monomethylsulochrin, and its molecular formula is C2. 18 H 18 O7, its chemical structural formula is shown below:

[0008] .

[0009] Monomethylsulfonamide is derived from the endophytic fungus Aspergillus fumigatus (… Aspergillus fumigatus Secondary metabolites of ).

[0010] Furthermore, the method for preparing monomethylsulfonamide includes the following steps:

[0011] (1) Fermentation of the strain: Pick a small amount of the strain with an inoculation loop Aspergillus fumigatus The mycelia (available at the China Microbial Culture Collection Center) were transferred to PDB liquid medium and cultured on a constant temperature shaker at 180 r / min and 28 ℃ for 3 days to obtain seed liquid. The seed liquid was then inoculated into sterilized rice medium using a sterile Pasteur pipette in a clean bench and fermented in the dark at 28 ℃ for 21 days.

[0012] (2) After the fermentation of the isolated strains, the substances in each culture medium were extracted three times with 400 mL of ethyl acetate by ultrasonic extraction, the residue was removed by vacuum filtration, and the organic solvent was evaporated by vacuum rotary evaporation at 40 °C to obtain a brown crude ethyl acetate phase extract. The crude extract was resuspended in distilled water and extracted three times in equal proportions with petroleum ether and ethyl acetate. After vacuum concentration, the degreased brown ethyl acetate residue was obtained.

[0013] (3) Ethyl acetate residues were subjected to normal silica gel column chromatography at 200-300 mesh with petroleum ether / ethyl acetate system as mobile phase, and gradient elution was performed with petroleum ether / ethyl acetate at a ratio of 90:10 to 10:90. Then, elution was performed with 100% methanol. The same components were identified and combined by TLC to obtain 16 secondary fractions Fr.1 to Fr.16.

[0014] (4) Fr.9 was subjected to normal silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase and gradient elution of 90:10 to 10:90 petroleum ether / ethyl acetate. The same components were identified and combined by TLC to obtain 9 secondary fractions Fr.9.1 to Fr.9.9. Fr.9.3 was subjected to reverse ODS column chromatography with methanol / water as the mobile phase and gradient elution of 20% to 100% methanol-water. The same components were identified and combined by TLC to obtain 11 tertiary fractions Fr.9.3.1 to Fr.9.3.11. Fr.9.3.1 was recrystallized to obtain the compound Monomethylsulochrin, i.e., monomethylsulfonylamine.

[0015] Furthermore, in step (1) of the above-mentioned method for preparing monomethylsulfonylpyrrolidone, the composition of the rice culture medium is: 70g rice + 110ml pure water per bottle of culture medium.

[0016] Furthermore, the above-mentioned use of monomethylsulfonylpyrrolidone in the preparation of a drug for treating ulcerative colitis, wherein the drug for treating ulcerative colitis further includes one or more pharmaceutically acceptable carriers or excipients.

[0017] Furthermore, the above-mentioned monomethylsulfonylamine is used in the preparation of a drug for treating ulcerative colitis, wherein the drug is prepared using monomethylsulfonylamine as the active ingredient and pharmaceutically acceptable excipients and processes.

[0018] Furthermore, the medication for treating ulcerative colitis is a drug that can improve symptoms such as diarrhea, rectal bleeding, and shortened colon length caused by ulcerative colitis.

[0019] Furthermore, the medication for treating ulcerative colitis is a drug that can improve the inflammatory response and / or pathological damage to colonic tissue caused by ulcerative colitis.

[0020] This invention investigates the effects of monomethylsulfonamide (MMSC) on ulcerative colitis using a DSS-induced acute ulcerative colitis model. ① The inflammatory characteristics of various mice were observed and compared. Results showed that colitis mice exhibited significant weight loss, severe diarrhea, rectal bleeding, and a significant shortening of the colon. In contrast, the weight loss in the MMSC treatment group was significantly inhibited, and diarrhea, bloody stools, and colon shortening were improved in a dose-dependent manner. This indicates that MMSC can alleviate inflammatory characteristics in UC mice. ② HE staining results showed that, compared with the DSS group, the ASA group and MMSC group showed significantly reduced inflammatory cell infiltration and tissue damage. This indicates that MMSC improved the histopathological damage in UC mice. ③ ELISA was used to detect the expression levels of four inflammatory factors in mouse colon tissue. Results showed that, compared with the Control group, the expression levels of IL-1β, IL-6, TNF-α, and IL-17 were significantly increased in the colon tissue of DSS-treated mice (P < 0.01); while compared with the DSS group, their expression levels were significantly downregulated in the ASA and MMSC groups (P < 0.01). This indicates that MMSCs can reduce the inflammatory response in UC mice.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention, through experimental research, has found that monomethylsulochrin can significantly improve DSS-induced weight loss, diarrhea, bloody stools, and shortened colon length in ulcerative colitis (UC) mice, alleviate inflammatory symptoms, effectively improve colonic tissue pathological damage, effectively inhibit the expression of inflammatory factors, and reduce the inflammatory response in UC mice. Experimental results indicate that monomethylsulochrin has a significant and definite therapeutic effect on ulcerative colitis and can be used to prepare drugs for treating ulcerative colitis.

[0023] This invention discloses for the first time the application of monomethylsulfonamide in the preparation of drugs for treating ulcerative colitis, providing a new drug source for the prevention and treatment of ulcerative colitis and broadening the application field of monomethylsulfonamide. Furthermore, monomethylsulfonamide has high safety, few toxic side effects, and significant therapeutic efficacy in treating ulcerative colitis, showing great promise for further development and utilization. Attached Figure Description

[0024] Figure 1 Inflammation characteristics in a UC mouse model; Figure 1In the diagram, A. Flowchart of the UC model creation experiment, B. Chemical structure diagram of MMSCs, C. Percentage change in body weight of UC mice, D. DAI index of mice in each group, E. Representative diagram of colon of mice in each group, F. Colon length of mice in each group, G. Thymus index of UC mice, H. Spleen index of UC mice (compared with the blank control group *P<0.05, **P<0.01, compared with the model control group #P<0.05, ##P<0.01).

[0025] Figure 2 HE staining of colon tissue from UC mice.

[0026] Figure 3 Expression of inflammatory factors in the colon tissue of UC mice; Figure 3 In the study, the levels of A. IL-1β in colon tissue; B. IL-6 in colon tissue; C. TNF-α in colon tissue; and D. IL-17 in colon tissue were compared with the blank control group (*P<0.05, **P<0.01; #P<0.05, ##P<0.01). Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials and reagents used are all commercially available. Example 1

[0028] I. Preparation of Monomethylsulfonamide

[0029] The preparation method of monomethylsulfonamide includes the following steps:

[0030] (1) Fermentation of the strain: Pick a small amount of the strain with an inoculation loop Aspergillus fumigatus The mycelia (purchased from the China Culture Collection Center for Microbial Cultures) were transferred to PDB liquid medium and cultured on a constant temperature shaker at 180 r / min and 28 ℃ for 3 days to obtain seed liquid. In a clean bench, the seed liquid was inoculated into sterilized rice medium (the composition of rice medium is: 70g rice + 110mL pure water per bottle) using a sterile Pasteur pipette and fermented in the dark at 28 ℃ for 21 days.

[0031] (2) After the fermentation of the isolated strain, the substance in each bottle of culture medium was extracted three times with 400 mL of ethyl acetate by ultrasonic extraction, the residue was removed by vacuum filtration, and the organic solvent was evaporated by vacuum rotary evaporation at 40 °C to obtain a brown crude ethyl acetate phase extract. The crude extract was resuspended in distilled water and extracted three times in equal proportions with petroleum ether and ethyl acetate. After vacuum concentration, the degreased brown ethyl acetate residue was obtained.

[0032] (3) Ethyl acetate residues were subjected to normal silica gel column chromatography (200-300 mesh) with petroleum ether / ethyl acetate system as mobile phase, and gradient elution was performed by (90:10-10:90) petroleum ether / ethyl acetate, followed by elution with 100% methanol. The same components were identified and combined by TLC to obtain 16 secondary fractions (Fr.1-Fr.16).

[0033] (4) Fr.9 was subjected to normal silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase and gradient elution of (90:10 to 10:90) petroleum ether / ethyl acetate. The same components were identified and combined by TLC to obtain 9 secondary fractions (Fr.9.1 to Fr.9.9); Fr.9.3 was subjected to ODS reverse column chromatography with methanol / water as the mobile phase and gradient elution of 20% to 100% methanol-water. The same components were identified and combined by TLC to obtain 11 tertiary fractions (Fr.9.3.1 to Fr.9.3.11). Fr.9.3.1 was recrystallized to obtain compound 1, namely compound Monomethylsulochrin.

[0034] Results: Compound structure identification: Compound 1, yellow-green crystals, HRESIMS m / z 369.0939 [M+Na] + 715.2095 [2M+Na] + The molecular formula is C 18 H 18 O7 has 10 degrees of unsaturation in its molecule. 1 H NMR (600 MHz, CD3OD) δ H 6.95 (1H, d, J =2.2 Hz, H-6'), 6.67 (1H, d, J =2.2 Hz, H-4'), 6.38 (1H, dd, J =1.5, 0.8 Hz, H-4), 6.20 (1H, s, H-2), 3.68 (3H, s, H-10), 3.65 (3H, s, H-8'), 3.37 (3H, s, H-9), 2.28 (3H, s, H-7). 13 C NMR (150 MHz, CD3OD) δ C201.61 (C, C-8), 167.96 (C, C-7′), 165.29 (C, C-5), 162.63 (C, C-1), 159.72 (C, C -5'), 158.48 (C, C-3'), 149.56 (C, C-3), 129.84 (C, C-1'), 128.05 (CH, C-2'), 111 0.75 (C, C-6), 111.38 (CH, C-4), 108.69 (CH, C-6'), 104.08 (CH, C-2), 104.06 (CH, C-4'), 56.52 (CH3, C-10), 56.25 (CH3, C-9), 52.54 (CH3, C-8'), 22.38 (CH3, C-7). The compound was identified as Monomethylsulochrin.

[0035] II. The therapeutic effect of monomethylsulfonamide (MMSC) on ulcerative colitis

[0036] method:

[0037] 1. DSS-induced acute UC model

[0038] Sixty male C57BL / 6 mice aged 6–8 weeks (20–22 g) were selected and fed acclimatizingly for 7 days. Then, the mice were randomly divided into 5 groups according to their body weight, with 12 mice in each group: normal group, model group, mesalazine group (200 mg / kg), low-dose MMSC administration group (60 mg / kg), and high-dose MMSC administration group (120 mg / kg).

[0039] For the first 6 days, except for the normal group mice which drank sterile water, the other groups of mice were given 2% DSS (sodium dextran sulfate) for free drinking to induce the UC mouse model. After the 6th day, all groups were switched to sterile water. While establishing the model, the corresponding drugs were administered by gavage at the same time every day at a dose of 0.1 mL / 10 g for 10 consecutive days. The specific dosage is shown in Table 1.

[0040] On day 11, tissue samples were collected from the mice. Serum, feces, and colon tissues were collected and stored at low temperature for further research.

[0041]

[0042] 2. Disease Activity Index (DAI) Assessment

[0043] Mice were weighed daily, and their fecal characteristics and bloody stools were observed. The DAI score was calculated according to the scoring criteria (Table 2). DAI = (Weight Loss Score + Fecal Characteristics Score + Bloody Score) / 3

[0044]

[0045] 3. Changes in colon length

[0046] On day 11, the mouse colon was harvested, its natural length (cm) was measured with a ruler, recorded, and photographed for record-keeping.

[0047] 4. Calculate organ indices by taking the spleen and thymus.

[0048] On day 11, the spleen and thymus of the mice were dissected and weighed using an electronic balance to calculate the organ index.

[0049] Spleen index = Spleen wet weight (mg) / Body weight (g);

[0050] Thymus index = thymus wet weight (mg) / body weight (g);

[0051] Liver index = Liver wet weight (mg) / Body weight (g);

[0052] Kidney index = Liver wet weight (mg) / Body weight (g).

[0053] 5. Colon tissue sections and HE staining

[0054] A portion of colonic tissue was extracted and fixed in 4% paraformaldehyde. After dehydration with alcohol and permeation with xylene, the tissue was embedded in paraffin and sectioned. The sections were stained with hematoxylin (H) and eosin (E), dehydrated and cleared again, and finally mounted with neutral resin. The pathological changes of colonic tissue, such as colonic congestion, ulceration, and intestinal wall thickening, were observed under an optical microscope, and the colonic tissue pathology was scored according to the scoring criteria in Table 3.

[0055]

[0056] 6. ELISA method for detecting the expression levels of inflammatory factors in colonic tissue

[0057] Mouse colon tissue was collected, and the expression levels of inflammatory factors such as TNF-α, IL-6, IL-17, and IL-1β were detected according to the ELISA kit instructions.

[0058] 7. Data Analysis and Statistics

[0059] All experimental data were summarized in Excel and analyzed using SPSS 21 software. Quantitative data that were normally distributed were described using the s-value; those that were not normally distributed were described using the M-value (P25–P27). Data that were both normally distributed and homogeneous in variance were compared between two groups using a t-test, and compared among multiple groups using one-way ANOVA. Ordinal data were described using the median and subjected to a rank-sum test. A p-value < 0.05 was considered statistically significant, and graphs were created using GraphPadPrism 9.5 software.

[0060] result:

[0061] 1. MMSCs improved DSS-induced ulcerative colitis

[0062] The ameliorative effect of MMSCs on UC mice was evaluated by the percentage of body weight loss, colon length, and DAI index. Figure 1 This is a diagram illustrating the inflammatory characteristics of a UC mouse model. Figure 1 middle, Figure 1 -A UC model creation experiment flowchart Figure 1 - B is the chemical structure of MMSC. Figure 1 -C is a line graph showing the change in body weight of UC mice. Figure 1 -D is the DAI exponent. (From...) Figure 1 -C indicates that the overall body weight of mice in the Control group did not change significantly; the weight of mice in the DSS group decreased significantly from the 4th day of modeling and continued to decrease as the experiment progressed (P < 0.01); compared with the DSS group, the weight loss of mice in the ASA group and MMSC group was less severe (P < 0.01). Figure 1 - D showed that the DAI index was lowest in the Control group and highest in the DSS group. Compared with the DSS group, the DAI index of the ASA group and MMSC group decreased significantly (P < 0.01). Figure 1 -E and Figure 1 -F represents the quantitative measurement of colon length in each group of mice. The results showed that the control group had the longest colon length, the DSS group had the shortest colon length, while the ASA and MMSC groups significantly inhibited colon shortening (P < 0.01). Figure 1 -G and Figure 1 -H represents the thymus and spleen indices in UC mice. The thymus and spleen are important immune organs, and the spleen and thymus indices are crucial indicators for assessing inflammation levels. The results showed that the DSS-induced ulcerative colitis model group mice exhibited significant splenomegaly and thymus atrophy, leading to an increased spleen index and a decreased thymus index. However, after administration of ASA and MMSCs, the spleen and thymus indices showed a trend towards returning to normal. Figure 1In a comparative study of inflammatory manifestations in various mice, mice with colitis showed significant weight loss, severe diarrhea, and rectal bleeding, along with a significant shortening of the colon. In contrast, the MMSC treatment group exhibited significantly inhibited weight loss and improved diarrhea, bloody stools, and colon shortening in a dose-dependent manner. This indicates that MMSCs can alleviate inflammatory manifestations in UC mice.

[0063] 2. MMSCs improved the histopathological damage in UC mice.

[0064] HE staining results showed that in the Control group mice, the colon cells were tightly packed, with abundant goblet cells and no obvious inflammatory infiltration or damage. In contrast, the DSS group mice showed extensive damage and edema to the colonic mucosa and tissues, a significant reduction in goblet cells, a loose structure, and severe lymphocyte infiltration. Compared with the DSS group, the ASA and MMSC groups showed significantly reduced inflammatory cell infiltration and tissue damage. HE staining images of UC mouse colon tissue are shown below. Figure 2 .

[0065] 3. MMSCs inhibited the expression of inflammatory mediators in UC mice.

[0066] The expression levels of four inflammatory factors in mouse colon tissue were detected using ELISA. Compared with the control group, the expression levels of IL-1β, IL-6, TNF-α, and IL-17 were significantly increased in the colon tissue of mice in the DSS group (P < 0.01). However, compared with the DSS group, their expression levels were significantly downregulated in the ASA and MMSC groups (P < 0.01). Figure 3 The expression of inflammatory factors in the colon tissue of UC mice. Figure 3 In the study, the levels of A. IL-1β in colon tissue; B. IL-6 in colon tissue; C. TNF-α in colon tissue; and D. IL-17 in colon tissue were compared with the blank control group (*P<0.05, **P<0.01; #P<0.05, ##P<0.01). Figure 3 Analysis showed that MMSCs could reduce the inflammatory response in UC mice.

[0067] in conclusion:

[0068] Monomethylsulfonamide has a significant and definite therapeutic effect on ulcerative colitis and can effectively treat ulcerative colitis.

Claims

1. The use of monomethylsulochrin in the preparation of drugs for treating ulcerative colitis, wherein the English name of monomethylsulochrin is: Monomethylsulochrin, and the molecular formula is C2. 18 H 18 O7, its chemical structural formula is shown below: 。 2. The application according to claim 1, characterized in that, The method for preparing monomethylsulfonamide includes the following steps: (1) Fermentation of the strain: a small amount of the strain was picked up with an inoculation loop. Aspergillus fumigatus The mycelia were transferred to PDB liquid medium and cultured on a constant temperature shaker at 180 r / min and 28 ℃ for 3 days to obtain seed liquid. In a clean bench, the seed liquid was inoculated into sterilized rice medium using a sterile Pasteur pipette and fermented in the dark at 28 ℃ for 21 days. (2) After the fermentation of the isolated strains, the substances in each culture medium were extracted three times with 400 mL of ethyl acetate by ultrasonic extraction, the residue was removed by vacuum filtration, and the organic solvent was evaporated by vacuum rotary evaporation at 40 °C to obtain a brown crude ethyl acetate phase extract. The crude extract was resuspended in distilled water and extracted three times in equal proportions with petroleum ether and ethyl acetate. After vacuum concentration, the degreased brown ethyl acetate residue was obtained. (3) Ethyl acetate residues were subjected to normal silica gel column chromatography at 200-300 mesh with petroleum ether / ethyl acetate system as mobile phase, and gradient elution was performed with petroleum ether / ethyl acetate at a ratio of 90:10 to 10:

90. Then, elution was performed with 100% methanol. The same components were identified and combined by TLC to obtain 16 secondary fractions Fr.1 to Fr.

16. (4) Fr.9 was subjected to normal silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase and gradient elution of 90:10 to 10:90 petroleum ether / ethyl acetate. The same components were identified and combined by TLC to obtain 9 secondary fractions Fr.9.1 to Fr.9.

9. Fr.9.3 was subjected to reverse ODS column chromatography with methanol / water as the mobile phase and gradient elution of 20% to 100% methanol-water. The same components were identified and combined by TLC to obtain 11 tertiary fractions Fr.9.3.1 to Fr.9.3.

11. Fr.9.3.1 was recrystallized to obtain the compound Monomethylsulochrin, i.e., monomethylsulfonylamine.

3. The application according to claim 2, characterized in that, In step (1) of the preparation method of monomethylsulfonamide, the composition of the rice culture medium is: 70g rice + 110mL pure water per bottle of culture medium.

4. The application according to claim 1, characterized in that, The medication for treating ulcerative colitis also includes one or more pharmaceutically acceptable carriers or excipients.

5. The application according to claim 1, characterized in that, The drug is prepared using monomethylsulfonylpyrrolidone as the active ingredient and pharmaceutically acceptable excipients and processes.

6. The application according to claim 1, characterized in that, The medication mentioned is for treating ulcerative colitis and is capable of improving symptoms such as diarrhea, rectal bleeding, and shortened colon length caused by ulcerative colitis.

7. The application according to claim 1, characterized in that, The medication for treating ulcerative colitis is one that can improve the inflammatory response and / or pathological damage to colonic tissue caused by ulcerative colitis.

Images