Application of monomethylsulfoline in preparation of medicine for treating ulcerative colitis

Abstract

The invention relates to the technical field of medicines, and particularly discloses application of monomethylsulfoline in preparation of a medicine for treating ulcerative colitis. Experimental research finds that Monomethylsulochrin can obviously improve the conditions of weight loss, diarrhea, hemafecia, shortened colon length and the like of a mouse with UC (ulcerative colitis) induced by DSS, relieve inflammatory characterization of the UC mouse, effectively improve pathological damage of colon tissue of the UC mouse, effectively inhibit expression of inflammatory factors of the UC mouse and relieve inflammatory response of the UC mouse. Researches show that the monomethylsulfoline has an obvious and exact effect on treating ulcerative colitis, and can be used for preparing the medicine for treating ulcerative colitis. The invention provides a new medicine source for preventing and treating ulcerative colitis, and widens the application field of methylsulfoline.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of monomethylsulfonylamine in preparing a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease that mainly affects the mucosal layer of the colon and rectum. It is characterized by inflammation and ulcers in the inner wall of the colon, leading to symptoms such as diarrhea, abdominal pain, and blood in the stool. The exact cause of the disease is still unclear, but it may be related to genetics, immune system abnormalities, and environmental factors. With the increasing incidence rate year by year, it has brought a great burden to global public health. At present, Western medicine treatments for ulcerative colitis mainly include salicylic acid preparations, immunosuppressants, glucocorticoids, and biological inhibitors, but they all have side effects such as large side effects and easy recurrence after discontinuation of medication. For example, 5-aminosalicylic acid drugs may cause loss of appetite, burning sensation in the stomach, nausea and vomiting. Long-term use of adrenal glucocorticoids may cause side effects such as osteoporosis and femoral head necrosis. Immunosuppressants and biological agents may bring risks such as hypertension, opportunistic infections, and nephrotoxicity. The relatively safe stem cell therapy is limited in clinical application due to its long treatment time and high price. Therefore, there is an urgent need to find safer, more effective, and less economical therapeutic drugs. Traditional Chinese medicine for the treatment of UC has the characteristics of flexible medication, multi-effects, less toxic and side effects, and low price, so it is worth investing efforts to explore its effectiveness and safety.

[0003] Natural products are a huge resource treasure trove for the development of new drugs. Their diverse structural types (including steroids, polyketides, non-ribosomal peptides, terpenes, alkaloids, etc.) and significant biological activities (including antioxidant, anti-inflammatory, anti-tumor, antibacterial, etc.) make them play an important role as lead drugs for the treatment of human diseases. Among them, the special ecological environment of the ocean has created a special metabolic mechanism for marine microorganisms, which in turn produces metabolites with novel structures and unique activities, and is an important source of new marine natural products. These compounds have significant pharmacological activities, such as anti-tumor and anti-inflammatory. Marine natural products have become one of the most promising new areas in the development of new drugs.

[0004] Tung Blossom Tree is one of the important tree species in mangrove forests. Studies have shown that Tung Blossom Tree has anti-inflammatory pharmacological activity. The compound Monomethylsulochrin (MMSC) is an endophytic fungus Aspergillus fumigatus ( Aspergillus fumigatus ) is a secondary metabolite with the molecular formula C 18 H 18O7, whose Chinese name is Monomethylsulochrin, belongs to type II polyketides. The main effects of type II polyketides include antibacterial, anti-tumor, immunosuppressive and anti-inflammatory. Existing studies on Monomethylsulochrin have shown that it has antibacterial and anti-glioma biological activities, but there are no reports on its effects on ulcerative colitis. Summary of the invention

[0005] The purpose of the present invention is to provide an application of monomethylsulochrin in the preparation of a drug for treating ulcerative colitis. The present invention discloses a new application of monomethylsulochrin, which provides a new drug source with high safety and good efficacy for the prevention and treatment of ulcerative colitis.

[0006] The technical solution adopted by the present invention is as follows: The application of monomethylsulochrin in the preparation of a drug for treating ulcerative colitis. The English name of the monomethylsulochrin is: Monomethylsulochrin, and the molecular formula is C 18 H 18 O7, its chemical structure is as follows: .

[0007] Monomethylsulfuron is derived from the endophytic fungus Aspergillus fumigatus ( Aspergillus fumigatus )'s secondary metabolites.

[0008] Furthermore, the preparation method of monomethylsulfonylamine comprises the following steps: (1) Strain fermentation: Use an inoculation loop to pick up a small amount of strain Aspergillus fumigatus The hyphae of the strain (available from China Microbiological Culture Collection Center) were transferred to PDB liquid culture medium, and cultured on a constant temperature shaker at 180 r / min and 28°C for 3 days to obtain seed liquid. In a clean bench, the seed liquid was inoculated into sterilized rice culture medium with a sterile Pasteur pipette, and fermented at 28°C in the dark for 21 days. (2) After the fermentation of the isolated strain is completed, the substances in each bottle of culture medium are ultrasonically extracted three times with 400 mL of ethyl acetate, the residue is removed by vacuum filtration, and the organic solvent is vacuum-rotated and evaporated at 40 °C to obtain a brown ethyl acetate phase crude extract. The crude extract is resuspended in distilled water, extracted three times with petroleum ether and ethyl acetate in equal proportions, and concentrated under reduced pressure to obtain a brown ethyl acetate residue after fat removal; (3) The ethyl acetate residue was subjected to normal flow silica gel column chromatography (200-300 mesh) with petroleum ether / ethyl acetate as the mobile phase, with a gradient elution of 90:10-10:90 petroleum ether / ethyl acetate, followed by elution with 100% methanol. The same components were combined by TLC to obtain 16 secondary fractions Fr.1-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 was performed from 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 was performed from 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.

[0009] Furthermore, in step (1) of the above-mentioned method for preparing monomethylsulfonylamine, the composition of the rice culture medium is: 70g rice + 110ml pure water per bottle of culture medium.

[0010] Furthermore, the above-mentioned monomethylsulfonylamine is used in the preparation of a drug for treating ulcerative colitis, wherein the drug for treating ulcerative colitis further comprises one or more pharmaceutically acceptable carriers or excipients.

[0011] Furthermore, the above-mentioned monomethylsulfonylamine is used in the preparation of a drug for treating ulcerative colitis. The drug is prepared with monomethylsulfonylamine as an active ingredient and using pharmaceutically acceptable excipients and processes.

[0012] Furthermore, the drug for treating ulcerative colitis is a drug that can improve the symptoms of diarrhea, bloody stools, and shortened colon length caused by ulcerative colitis.

[0013] Furthermore, the drug for treating ulcerative colitis is a drug that can improve the inflammatory response and / or pathological damage of colon tissue caused by ulcerative colitis.

[0014] The present invention uses DSS to induce an acute UC model to study the effect of monomethylsulfonyl chloramphenicol (MMSC) on ulcerative colitis. ① The experimental observations compared the inflammatory manifestations of various mice. The results showed that the weight of colitis mice was significantly reduced, and severe diarrhea and rectal bleeding occurred, and the length of the colon was significantly shortened; while the weight loss of mice in the MMSC treatment group was significantly inhibited, and diarrhea, bloody stools and colon shortening trends were improved in a dose-dependent manner. It shows that MMSC can alleviate the inflammatory manifestations of UC mice. ② The results of the HE staining experiment showed that compared with the DSS group, the infiltration of inflammatory cells and the degree of tissue damage in the ASA group and the MMSC group were significantly reduced. It shows that MMSC improves the tissue pathological damage of UC mice. ③ The expression levels of four inflammatory factors in the colon tissue of mice were detected by ELISA. The results showed that compared with the Control group, the expression levels of IL-1β, IL-6, TNF-a and IL-17 inflammatory factors were significantly increased in the colon tissue of the DSS group mice (P < 0.01); and compared with the DSS group, their expression levels were significantly downregulated in the ASA and MMSC groups (P < 0.01). This indicates that MMSC can alleviate the inflammatory response in UC mice.

[0015] The beneficial effects of the present invention are: The present invention has found through experimental research that monomethylsulochrin can significantly improve the weight loss, diarrhea, blood in stool, shortened colon length and other conditions of UC mice induced by DSS, reduce the inflammatory manifestations of UC mice, effectively improve the pathological damage of colon tissues of UC mice, effectively inhibit the expression of inflammatory factors in UC mice, and reduce the inflammatory response of UC mice. The experimental results show that monomethylsulochrin has an obvious and definite effect of treating ulcerative colitis and can be used to prepare a drug for treating ulcerative colitis.

[0016] The present invention discloses for the first time the use of monomethylsulfonylamine in the preparation of a drug for treating ulcerative colitis, provides a new drug source for the prevention and treatment of ulcerative colitis, and broadens the application field of mesosulfonylamine. In addition, monomethylsulfonylamine has high safety, low toxicity and side effects, and significant efficacy in treating ulcerative colitis, and has a good prospect for development and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Characterization of inflammation in the UC mouse model; Figure 1A. UC model experiment flow chart, B. Chemical structure of MMSC, C. Percentage of weight change of UC mice, D. DAI index of mice in each group, E. Representative 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 blank control group *P<0.05, **P<0.01, compared with model control group #P<0.05, ##P<0.01).

[0018] Figure 2 HE staining of colon tissue of UC mice.

[0019] Figure 3 Expression of inflammatory factors in colonic tissue of UC mice; Figure 3 A. IL-1β level in colon tissue; B. IL-6 level in colon tissue; C. TNF-α level in colon tissue; D. IL-17 level in colon tissue (compared with blank control group*P<0.05, **P<0.01, compared with model control group#P<0.05, ##P<0.01). DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the examples and drawings. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are all commercially available. Example 1

[0021] 1. Preparation of Monomethylsulfonylamine The preparation method of monomethylsulfonylamine comprises the following steps: (1) Strain fermentation: Use an inoculation loop to pick up a small amount of strain Aspergillus fumigatus The mycelium (purchased from China Microorganism Culture Collection Center) was transferred to PDB liquid culture medium and cultured on a constant temperature shaker at 180 r / min and 28 ℃ for 3 days to obtain seed solution. In an ultra-clean workbench, the seed solution was aspirated with a sterile Pasteur pipette and inoculated into sterilized rice culture medium (the composition of rice culture medium is: 70 g rice + 110 mL pure water per bottle of culture medium), and fermented at 28 ℃ in the dark for 21 days.

[0022] (2) Extraction and separation of strains After fermentation, the substances in each bottle of culture medium were ultrasonically extracted three times with 400 mL of ethyl acetate, and the residue was removed by vacuum filtration. The organic solvent was vacuum-rotated and evaporated at 40 °C to obtain a brown ethyl acetate phase crude extract. The crude extract was resuspended in distilled water and extracted three times with petroleum ether and ethyl acetate in equal proportions. After reduced pressure concentration, a brown ethyl acetate residue after fat removal was obtained.

[0023] (3) The ethyl acetate residue was subjected to normal silica gel column chromatography (200-300 mesh) with petroleum ether / ethyl acetate as the mobile phase, with a gradient elution of (90:10-10:90) petroleum ether / ethyl acetate, followed by elution with 100% methanol. The same components were combined by TLC to obtain 16 secondary fractions (Fr.1-Fr.16).

[0024] (4) Fr.9 was subjected to normal silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase, and gradient elution was performed from petroleum ether / ethyl acetate (90:10 to 10:90). 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 was performed from 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, i.e., compound Monomethylsulochrin.

[0025] Results: Compound structure identification Compound 1, yellow-green crystals, HRESIMS m / z :369.0939 [M+Na] + ,715.2095 [2M+Na] + , molecular formula is C 18 H 18 O7, contains 10 degrees of unsaturation in the 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 .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.

[0026] 2. Effect of MMSC in the treatment of ulcerative colitis method: 1. DSS-induced acute UC model Sixty male C57BL / 6 mice aged 6-8 weeks (20-22 g) were selected. After 7 days of adaptive feeding, they were randomly divided into 5 groups according to their body weight, with 12 mice in each group, namely normal group, model group, mesalazine group (200 mg / kg), MMSC low-dose group (60 mg / kg), and MMSC high-dose group (120 mg / kg).

[0027] In the first 6 days, except for the mice in the normal group who drank sterile water, the mice in the other groups were given 2% DSS (dextran sulfate sodium) to drink freely to induce the UC mouse model. After the 6th day, all groups were replaced with sterile water. While establishing the model, the corresponding drugs were gavaged at the same time every day at a dosage volume of 0.1 mL / 10 g for 10 consecutive days. The specific dosage is shown in Table 1.

[0028] On the 11th day, the mice were subjected to the experiment. Serum, feces, colon and other tissues of the mice were collected and stored at low temperature for further study.

[0029]

[0030] 2. Disease Activity Index (DAI) Assessment Weigh the mice daily, observe their stool characteristics and blood in stool, and calculate the DAI score of the mice according to the scoring criteria (Table 2). DAI = (weight loss score + stool characteristics score + stool bleeding score) / 3

[0031] 3. Changes in the length of the colon On the 11th day, the mouse colon was taken, and its natural length (cm) was measured with a ruler and recorded and photographed for preservation.

[0032] 4. Take the spleen and thymus to calculate the organ index.

[0033] On the 11th day, the spleen and thymus of the mice were dissected and weighed using an electronic balance to calculate the organ index.

[0034] Spleen index = spleen wet weight (mg) / body weight (g); Thymus index = thymus wet weight (mg) / body weight (g); Liver index = liver wet weight (mg) / body weight (g); Renal index = liver wet weight (mg) / body weight (g).

[0035] 5. Colon tissue sectioning and HE staining Part of the colon tissue was removed and fixed in 4% paraformaldehyde. After the tissue was dehydrated with alcohol and permeabilized with xylene, it was embedded in paraffin and sliced. The slices were stained with hematoxylin (H) and eosin (Eosin, E), dehydrated again to make them transparent, and finally sealed with neutral gum. The pathological changes of colon tissue such as colon congestion, ulceration, and intestinal wall thickening were observed under an optical microscope, and the colon tissue pathology was scored according to the scoring criteria in Table 3.

[0036]

[0037] 6. ELISA method to detect the expression level of inflammatory factors in colon tissue The colon tissues of mice were obtained, and the expression levels of inflammatory factors such as TNF-α, IL-6, IL-17 and IL-1β were detected according to the instructions of the ELISA kit.

[0038] 7. Data Analysis and Statistics All experimental data were summarized in Excel and analyzed using SPSS21 software. If the measurement data satisfied the normal distribution, s was used to describe it; if it did not satisfy the normal distribution, M (P25-P27) was used to describe it; for data that satisfied both the normal distribution and the homogeneity of variance, the t test was used to compare two groups, and the one-way analysis of variance was used to compare multiple groups; for the rank data, the median was used to describe it, and the rank sum test was performed; P < 0.05 indicated that the difference was statistically significant, and GraphPadPrism9.5 software was used to draw the graph.

[0039] result: 1. MMSCs improved DSS-induced ulcerative colitis The improvement effect of MMSC on UC mice was evaluated by weight loss percentage, colon length and DAI index. Figure 1This is a picture showing inflammation in the UC mouse model. Figure 1 middle, Figure 1 -A UC model modeling experimental process diagram, Figure 1 - B is the chemical structure of MMSC. Figure 1 -C is the line graph of weight change ratio of UC mice, Figure 1 -D is the DAI index. Figure 1 -C shows that the overall body weight of mice in the Control group did not change much; the weight of mice in the DSS group began to decrease significantly from the 4th day of modeling, and the weight 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 the MMSC group was less (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 the MMSC group decreased significantly (P<0.01). Figure 1 -E and Figure 1 -F is the quantification of colon length of each group of mice. The results showed that the colon length of the Control group mice was the longest, the colon of the DSS group was the shortest, and the shortening of the colon was significantly inhibited in the ASA and MMSC groups (P < 0.01). Figure 1 -G and Figure 1 -H is the thymus and spleen index of UC mice. The thymus and spleen are important immune organs of the body, and the spleen index and thymus index are important indicators for judging the level of inflammation. The results showed that the DSS-induced ulcerative colitis mice in the model group showed obvious splenomegaly and thymus atrophy, resulting in an increase in the spleen index and a decrease in the thymus index. After the administration of ASA and MMSC, the spleen and thymus indexes showed a trend of returning to normal. Figure 1 , the inflammatory signs of various mice were observed and compared. The colitis mice lost weight significantly and had severe diarrhea and rectal bleeding. The length of the colon was significantly shortened. The weight loss of mice in the MMSC treatment group was significantly inhibited, and diarrhea, bloody stools and colon shortening trends were improved in a dose-dependent manner. This shows that MMSC can alleviate the inflammatory signs of UC mice.

[0040] 2. MMSCs improved tissue pathological damage in UC mice HE staining results showed that the colon cells of the mice in the Control group were closely arranged, the number of goblet cells was abundant, and no obvious inflammatory infiltration or damage was observed. However, the colon mucosal epithelium and tissues of the mice in the DSS group were extensively damaged and edematous, the goblet cells were greatly reduced, the structure was loose, and there was severe lymphocyte infiltration. Compared with the DSS group, the degree of inflammatory cell infiltration and tissue damage in the ASA group and the MMSC group was significantly reduced. HE staining of the colon tissue of UC mice is shown in Figure 2. Figure 2 .

[0041] 3. MMSC inhibited the expression of inflammatory mediators in UC mice ELISA was used to detect the expression levels of four inflammatory factors in the colon tissue of mice. Compared with the Control group, the expression levels of IL-1β, IL-6, TNF-a and IL-17 inflammatory factors in the colon tissue of mice in the DSS group were significantly increased (P < 0.01). 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 colon tissue of UC mice. Figure 3 A. IL-1β level in colon tissue; B. IL-6 level in colon tissue; C. TNF-α level in colon tissue; D. IL-17 level in colon tissue (compared with blank control group*P<0.05, **P<0.01, compared with model control group#P<0.05, ##P<0.01). Figure 3 The analysis showed that MMSCs could alleviate the inflammatory response in UC mice.

[0042] in conclusion: Monomethylsulfonylamine has an obvious and definite effect in treating ulcerative colitis and can effectively treat ulcerative colitis.

Claims

1. The use of monomethylsulochrin in the preparation of a drug for treating ulcerative colitis, wherein the English name of the monomethylsulochrin is: Monomethylsulochrin, and the molecular formula is C 18 H 18 O7, its chemical structure is as follows: 。 2. The use according to claim 1, characterized in that: The preparation method of monomethylsulfonylamine comprises the following steps: (1) Strain fermentation: Use an inoculation loop to pick up a small amount of strain Aspergillus fumigatus The hyphae 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 with a sterile Pasteur pipette and fermented at 28 ℃ in the dark for 21 days. (2) After the fermentation of the isolated strain is completed, the substances in each bottle of culture medium are ultrasonically extracted three times with 400 mL of ethyl acetate, the residue is removed by vacuum filtration, and the organic solvent is vacuum-rotated and evaporated at 40 °C to obtain a brown ethyl acetate phase crude extract. The crude extract is resuspended in distilled water, extracted three times with petroleum ether and ethyl acetate in equal proportions, and concentrated under reduced pressure to obtain a brown ethyl acetate residue after fat removal; (3) The ethyl acetate residue was subjected to normal flow silica gel column chromatography (200-300 mesh) with petroleum ether / ethyl acetate as the mobile phase, with a gradient elution of 90:10-10:90 petroleum ether / ethyl acetate, followed by elution with 100% methanol. The same components were combined by TLC to obtain 16 secondary fractions Fr.1-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 was performed from 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 was performed from 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.

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

4. The use according to claim 1, characterized in that: The drug for treating ulcerative colitis further comprises one or more pharmaceutically acceptable carriers or excipients.

5. The use according to claim 1, characterized in that: The medicine is prepared by using monomethylsulfonylline as an effective ingredient and using pharmaceutically acceptable auxiliary materials and processes.

6. The use according to claim 1, characterized in that: The drug for treating ulcerative colitis is a drug that can improve the symptoms of diarrhea, blood in the stool and shortened colon length caused by ulcerative colitis.

7. The use according to claim 1, characterized in that: The drug for treating ulcerative colitis is a drug that can improve the inflammatory response and / or pathological damage of colon tissue caused by ulcerative colitis.

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