Use of norathyriol for the preparation of a medicament for the treatment of an inflammatory disease

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of Norathyriol in preparation of a medicine for treating inflammatory diseases. The application shows that Norathyriol has a binding capacity with NLRP3 inflammasome, Norathyriol can inhibit the expression of NLRP3, and then reduce the expression of IL-1beta gene in the downstream, so as to significantly reduce the secretion of IL-1beta in mouse bone marrow macrophages induced by LPS and Nigericin in a concentration-dependent manner. In an ulcerative colitis model, Norathyriol effectively reduces the DAI score, relieves weight loss, colon shortening and intestinal inflammation, and simultaneously significantly improves the pathological damage of colon tissue, including inflammatory cell infiltration, epithelial shedding and crypt structure destruction. In addition, Norathyriol also shows a significant therapeutic effect on a pneumonia model, reduces pulmonary edema and alveolar structure damage, improves weight loss and T cell differentiation imbalance caused by H1N1 infection, and improves organ indexes, indicating that Norathyriol has a dual effect of relieving lung inflammation and regulating immune function.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of Norathyriol in the preparation of drugs for treating inflammatory diseases. Background Technology

[0002] Inflammatory diseases are a significant area of ​​research in current medicine, with conditions such as ulcerative colitis and pleurisy caused by influenza A virus posing serious threats to human health. The pathogenesis of these inflammatory diseases is complex, primarily related to abnormal activation of the innate immune system. Research indicates that the NLRP3 inflammasome, as an important component of the host's innate immune system, plays a crucial role in inflammatory responses. The NLRP3 inflammasome is a polyprotein complex composed of the innate immune receptor NLRP3, the adaptor protein ASC, and caspase-1 precursors. Its assembly leads to caspase-1 activation, which subsequently promotes the cleavage of IL-1β and IL-18 precursors, resulting in mature, functional IL-1β and IL-18. Current research has shown that abnormal activation of the NLRP3 inflammasome is associated with a variety of diseases, including inflammatory diseases of the digestive tract (ulcerative colitis), central nervous system diseases (Parkinson's disease, Alzheimer's disease), obesity and obesity-related metabolic syndrome (type 2 diabetes), respiratory inflammatory diseases, and other inflammatory diseases. Therefore, the NLPR3 inflammasome is considered a potential drug target for treating inflammatory diseases.

[0003] CN116726003A discloses the application of eugenolactone in the preparation of drugs for the prevention and treatment of NLRP3-mediated diseases. This patent describes how Hel directly covalently binds to NLRP3 via a carbon-carbon double bond, interfering with the interaction between NLRP3 and NEK7, thereby inhibiting the activation of the NLRP3 inflammasome. Furthermore, Hel has shown significant therapeutic effects in several mouse models of NLRP3-mediated diseases.

[0004] CN116473966A discloses the application of SB-222200 in inhibiting NLRP3 inflammasome activation. This patent inhibits the assembly of the NLRP3 inflammasome by directly binding to the NLRP3 protein, and also inhibits the oligomerization and speckle formation of ASC protein during NLRP3 inflammasome activation, inhibits GSDMD cleavage and its pyroptosis, inhibits the oligomerization of the NLRP3 protein itself, and the interaction between the NLRP3 protein and ASC protein. Simultaneously, SB-222200 can also alleviate peritonitis and inflammatory bowel disease.

[0005] Patent CN113288936A discloses a pharmaceutical composition for treating ulcerative colitis and its application, the composition comprising a feces extract. This patent demonstrates the therapeutic effects of this pharmaceutical composition in alleviating weight loss, reducing DAI scores, lengthening colon length, improving colonic gross morphology scores, reducing inflammatory infiltration, and inhibiting the accumulation of MPO enzymes in colonic tissue in mice with DSS-induced ulcerative colitis. However, this patent still faces the challenge of further screening and optimizing the preparation methods of the feces extract and gardenia extract to improve the therapeutic efficacy and bioavailability of the pharmaceutical composition.

[0006] Patent CN116920012A relates to the application of the blood-stopping extract in the preparation of a drug for treating ulcerative colitis. This patent demonstrates that the blood-stopping extract can effectively reduce DAI scores, inhibit plasma inflammatory factor levels, alleviate inflammatory responses in colonic mucosa, and exert a protective effect on the colonic mucosa. However, this patent still requires further investigation into the optimal dosage and administration method of the blood-stopping extract in the treatment of ulcerative colitis to improve the therapeutic efficacy and safety of the drug.

[0007] Norathyriol is a deglycosylated product of mangiferin, belonging to the flavonoid class of compounds, and possesses various biological activities including antioxidant, anti-inflammatory, and antitumor effects. Its structural characteristics make it more lipid-soluble than mangiferin, which may affect its absorption and mechanism of action in vivo. As a natural compound with potential medicinal value, Norathyriol shows promise for development in functional foods and pharmaceutical research.

[0008] However, the application of Norathyriol in the preparation of drugs for treating inflammatory diseases has not been fully developed and utilized, especially in the treatment of inflammatory diseases caused by abnormal activation of the NLRP3 inflammasome, such as ulcerative colitis, influenza A virus-induced inflammation, or pleurisy. The application value of Norathyriol needs to be explored in depth. Summary of the Invention

[0009] To address the aforementioned shortcomings, this invention proposes for the first time the unique therapeutic effects of Norathyriol in the treatment of inflammatory diseases. Experimental studies have shown that Norathyriol exerts its therapeutic effects through multiple targets and pathways. This invention provides a new option for the clinical treatment of inflammatory diseases and has significant clinical application value.

[0010] The technical solution of this invention is as follows:

[0011] On the one hand, this invention provides the application of Norathyriol in the preparation of medicaments for treating inflammatory diseases.

[0012] Specifically, the structural formula of Norathyriol is:

[0013]

[0014] Specifically, the inflammatory disease is mediated by abnormal activation of the NLRP3 inflammasome.

[0015] Preferably, the inflammatory disease is selected from:

[0016] (a) Inflammatory diseases of the digestive tract;

[0017] (b) Inflammatory diseases of the respiratory tract.

[0018] Preferably, the inflammatory disease of the digestive tract is ulcerative colitis; the inflammatory disease of the respiratory tract is pleurisy or inflammation caused by influenza A virus.

[0019] Preferably, the ulcerative colitis includes, but is not limited to, acute ulcerative colitis or chronic ulcerative colitis.

[0020] Preferably, the influenza A virus can be the H1N1 virus.

[0021] Specifically, Norathyriol is the sole or primary active ingredient of the drug.

[0022] Preferably, the content of Norathyriol in the drug may be greater than 85%.

[0023] Preferably, the content of Norathyriol in the drug can be 99.83%.

[0024] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0025] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0026] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0027] Specifically, the dosage form of the drug is an injection, capsule, tablet, powder, granule, pill, microcapsule / microsphere preparation, suppository, ointment, spray, or targeted preparation.

[0028] Specifically, the administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0029] Preferably, the drug can be administered orally.

[0030] Specifically, the drug can also be used in combination with other drugs for the prevention and / or treatment of ulcerative colitis.

[0031] More specifically, the combined use includes sequential or simultaneous use.

[0032] In another aspect, the present invention provides the use of isomers of Norathyriol in the preparation of medicaments for treating inflammatory diseases.

[0033] Specifically, the isomer may be a positional isomer.

[0034] Specifically, the inflammatory disease is mediated by abnormal activation of the NLRP3 inflammasome.

[0035] Preferably, the inflammatory disease is selected from:

[0036] (a) Inflammatory diseases of the digestive tract;

[0037] (b) Inflammatory diseases of the respiratory tract;

[0038] (c) Other diseases mediated by abnormal activation of the NLRP3 inflammasome.

[0039] Preferably, the inflammatory disease of the digestive tract is ulcerative colitis; the inflammatory disease of the respiratory tract is pleurisy or inflammation caused by influenza A virus.

[0040] Preferably, the ulcerative colitis includes, but is not limited to, acute ulcerative colitis or chronic ulcerative colitis.

[0041] Preferably, the influenza A virus can be the H1N1 virus.

[0042] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0043] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0044] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0045] Specifically, the dosage form of the drug is an injection, capsule, tablet, powder, granule, pill, microcapsule / microsphere preparation, suppository, ointment, spray, or targeted preparation.

[0046] Specifically, the administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0047] Preferably, the drug can be administered orally.

[0048] In another aspect, the present invention provides the use of solvates of Norathyriol in the preparation of medicaments for treating inflammatory diseases.

[0049] Specifically, the solvates include, but are not limited to, hydrates, methanol compounds, ethanol compounds, isopropanol compounds, or DMSO compounds.

[0050] Specifically, the inflammatory disease is mediated by abnormal activation of the NLRP3 inflammasome.

[0051] Preferably, the inflammatory disease is selected from:

[0052] (a) Inflammatory diseases of the digestive tract;

[0053] (b) Inflammatory diseases of the respiratory tract;

[0054] (c) Other diseases mediated by abnormal activation of the NLRP3 inflammasome.

[0055] Preferably, the inflammatory disease of the digestive tract is ulcerative colitis; the inflammatory disease of the respiratory tract is pleurisy or inflammation caused by influenza A virus.

[0056] Preferably, the ulcerative colitis includes, but is not limited to, acute ulcerative colitis or chronic ulcerative colitis.

[0057] Preferably, the influenza A virus can be the H1N1 virus.

[0058] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0059] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0060] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0061] Specifically, the dosage form of the drug is an injection, capsule, tablet, powder, granule, pill, microcapsule / microsphere preparation, suppository, ointment, spray, or targeted preparation.

[0062] Specifically, the administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0063] Preferably, the drug can be administered orally.

[0064] In another aspect, the present invention provides the use of pharmaceutically acceptable salts of Norathyriol in the preparation of medicaments for treating inflammatory diseases.

[0065] Specifically, the pharmaceutically acceptable salts include, but are not limited to, sodium salts, potassium salts, meglumine salts, zinc complexes, or iron complexes.

[0066] Specifically, the inflammatory disease is mediated by abnormal activation of the NLRP3 inflammasome.

[0067] Preferably, the inflammatory disease is selected from:

[0068] (a) Inflammatory diseases of the digestive tract;

[0069] (b) Inflammatory diseases of the respiratory tract;

[0070] (c) Other diseases mediated by abnormal activation of the NLRP3 inflammasome.

[0071] Preferably, the inflammatory disease of the digestive tract is ulcerative colitis; the inflammatory disease of the respiratory tract is pleurisy or inflammation caused by influenza A virus.

[0072] Preferably, the ulcerative colitis includes, but is not limited to, acute ulcerative colitis or chronic ulcerative colitis.

[0073] Preferably, the influenza A virus can be the H1N1 virus.

[0074] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0075] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0076] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0077] Specifically, the dosage form of the drug is an injection, capsule, tablet, powder, granule, pill, microcapsule / microsphere preparation, suppository, ointment, spray, or targeted preparation.

[0078] Specifically, the administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0079] Preferably, the drug can be administered orally.

[0080] In another aspect, the present invention provides the application of Norathyriol cocrystals in the preparation of medicaments for treating inflammatory diseases.

[0081] Specifically, the eutectic forming agent of the eutectic can be selected from one or more of oxalic acid, citric acid, succinic acid, nicotinamide, urea, caffeine, isonicotinamide, glucose or lactose.

[0082] Specifically, the inflammatory disease is mediated by abnormal activation of the NLRP3 inflammasome.

[0083] Preferably, the inflammatory disease is selected from:

[0084] (a) Inflammatory diseases of the digestive tract;

[0085] (b) Inflammatory diseases of the respiratory tract;

[0086] (c) Other diseases mediated by abnormal activation of the NLRP3 inflammasome.

[0087] Preferably, the inflammatory disease of the digestive tract is ulcerative colitis; the inflammatory disease of the respiratory tract is pleurisy or inflammation caused by influenza A virus.

[0088] Preferably, the ulcerative colitis includes, but is not limited to, acute ulcerative colitis or chronic ulcerative colitis.

[0089] Preferably, the influenza A virus can be the H1N1 virus.

[0090] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0091] Preferably, the pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0092] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0093] Specifically, the dosage form of the drug is an injection, capsule, tablet, powder, granule, pill, microcapsule / microsphere preparation, suppository, ointment, spray, or targeted preparation.

[0094] Specifically, the administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0095] Preferably, the drug can be administered orally.

[0096] The beneficial effects of this invention are as follows:

[0097] (1) Norathyriol has a strong direct binding ability to the NLRP3 inflammasome. Norathyriol can inhibit the secretion of IL-1β in mouse bone marrow macrophages induced by LPS and Nigericin. The mechanism may be through inhibiting NLRP3 expression, thereby reducing the expression of its downstream IL-1β gene, and this inhibitory effect is concentration-dependent.

[0098] (2) Norathyriol improves the DAI score of DSS-induced ulcerative colitis; Norathyriol improves the weight loss caused by DSS-induced ulcerative colitis, and its effect is better than that of the marketed drug mesalazine; Norathyriol can significantly reduce the increase in inflammation caused by ulcerative colitis and has a significant therapeutic effect on improving intestinal inflammation; Norathyriol improves the shortening of the colon caused by DSS-induced ulcerative colitis, and its effect is better than that of the marketed drug mesalazine; Norathyriol reduces inflammatory cell infiltration, epithelial shedding and crypt structure destruction in colonic tissue.

[0099] (3) Norathyriol improves pulmonary edema caused by pneumonia and increases the indices of other organs, revealing its role in relieving pulmonary inflammation and improving immune status; Norathyriol improves weight loss caused by H1N1 infection; Norathyriol significantly improves the imbalance of T cell differentiation caused by H1N1 infection and has a good therapeutic effect on pneumonia; Norathyriol improves alveolar wall thickening and inflammatory infiltration, and relieves alveolar expansion and rupture. Attached Figure Description

[0100] Figure 1 This is an analytical diagram of the SPR sensor image in Example 1.

[0101] Figure 2 In Example 1, the IL-1β content in the supernatant was detected by ELISA. In the figure, "*" represents p < 0.05 compared with the LPS+Nigericin stimulation group only; "**" represents p < 0.01 compared with the LPS+Nigericin stimulation group only; and "***" represents p < 0.001 compared with the LPS+Nigericin stimulation group only.

[0102] Figure 3 The figure shows the relative expression level of the NLRP3 gene detected by real-time fluorescence quantitative PCR in Example 1. The "***" in the figure represents p < 0.001 compared with the LPS+Nigericin stimulation group only.

[0103] Figure 4 The figure shows the relative expression level of the IL-1β gene detected by real-time fluorescence quantitative PCR in Example 1. The “****” in the figure represents p < 0.0001 compared with the LPS+Nigericin stimulation group only.

[0104] Figure 5 The graph shows the DAI score results for each group in Example 2.

[0105] Figure 6 This is a graph showing the trend of weight change in each group in Example 2.

[0106] Figure 7 The figure shows the IL-1β content in the serum of mice in each group in Example 2. In the figure, "*" represents p < 0.05 compared with the model group; "**" represents p < 0.01 compared with the model group; and "###" represents p < 0.001 compared with the blank group.

[0107] Figure 8 The image shows the colon length results for each group in Example 2.

[0108] Figure 9 These are histopathological images of each group in Example 2.

[0109] Figure 10The graph shows the ratios of lung tissue, spleen tissue, and thymus tissue to body weight in Example 3. In the graph, "*" represents p < 0.05; "***" represents p < 0.001; and "****" represents p < 0.0001.

[0110] Figure 11 This is a graph showing the average change in body weight of mice in each group in Example 3.

[0111] Figure 12 The image shows a flow cytometry plot of CD4+ T cells in the blank lung tissue group in Example 3.

[0112] Figure 13 The image shows a flow cytometry plot of CD4+ T cells from the lung tissue model group in Example 3.

[0113] Figure 14 The image shows a flow cytometry plot of CD4+ T cells in the lung tissue positive drug group in Example 3.

[0114] Figure 15 The image shows a flow cytometry plot of CD4+ T cells in the Norathyriol lung tissue group from Example 3.

[0115] Figure 16 The image shows a flow cytometry plot of CD8+ T cells in the blank lung tissue group in Example 3.

[0116] Figure 17 The image shows a flow cytometry plot of CD8+ T cells from the lung tissue model group in Example 3.

[0117] Figure 18 The image shows a flow cytometry plot of CD8+ T cells in the lung tissue positive drug group in Example 3.

[0118] Figure 19 The image shows a flow cytometry plot of CD8+ T cells in the Norathyriol lung tissue group from Example 3.

[0119] Figure 20 This is a pathological image of lung tissue from Example 3. Detailed Implementation

[0120] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0121] The structural formula of Norathyriol (MedChemExpress, HY-N1029) described in this invention is as follows:

[0122]

[0123] Example 1: Regulatory effect of Norathyriol on the activation mechanism of NLRP3 inflammasome

[0124] 1.1 Method for detecting the binding activity of Norathyriol to NLRP3 protein using the Biacore 8K platform

[0125] (1) Experimental methods

[0126] In this experiment, a CM5 chip was used. Recombinant and purified NLRP3 protein was immobilized on the chip surface using an EDC / NHS chemical coupling method. The coupling buffer was a 10 mM sodium acetate buffer at pH 5.5. After immobilization, different concentrations (e.g., 0.156–10 μM) of Norathyriol solution were sequentially injected into the chip using HBS-EP+ buffer containing 2% DMSO at a flow rate of 30 μL / min. The binding time was 120 seconds, and the dissociation time was 300 seconds. Experimental data were processed using Biacore 8K Evaluation software. After subtracting the reference signal from the blank channel, a 1:1 Langmuir model was used to calculate the binding affinity of Norathyriol to NLRP3 protein.

[0127] (2) Results

[0128] The results are shown in Table 1:

[0129] Table 1

[0130] 1.94 x 10 2 ]] 4.10 x 10 -5 ]] 186.1 7.5

[0131] The results are as follows Figure 1 As shown in Table 1, the interaction between Norathyriol and NLRP3 protein was detected using the Biacore 8K surface plasmon resonance (SPR) system. The affinity constant (KD) was measured to be 41 μM, indicating that there is a strong affinity between the two proteins.

[0132] 1.2 Effects of Norathyriol on IL-1β secretion and gene expression in an LPS / Nigericin-induced BMDMs inflammation model

[0133] This embodiment mainly studies the effects of LPS (lipopolysaccharide), Nigericin, and Norathyriol on IL-1β secretion and NLRP3 and IL-1β gene expression in mouse bone marrow macrophages (BMDMs).

[0134] (1) Experimental method:

[0135] Mouse bone marrow macrophages (BMDMs) were differentiated for 7 days in RPMI-1640 medium supplemented with 20 ng / mL M-CSF. The cells were 1 × 10⁻⁶ cells per ... 5 Cells were seeded per well in 12-well plates. After 24 h of adhesion, the medium was replaced with fresh medium containing 50 ng / mL LPS and specified drug concentrations (0 μM, 5 μM, 10 μM, 20 μM, and 40 μM Norathyriol) and cultured for 3 h. Cells were then stimulated with 10 μM Nigericin for 30 min. The IL-1β content in the supernatant was then measured using an ELISA kit (STARTER, S0C3029). Total RNA was extracted from the cells using the Trizol method, and the intracellular NLRP3 and IL-1β gene expression levels were measured using real-time quantitative PCR.

[0136] (2) Experimental Results

[0137] Depend on Figure 2 The results showed that the addition of norathyriol decreased IL-1β secretion in a concentration-dependent manner. Statistical analysis indicated that a significant difference was observed at a norathyriol concentration of 10 μM (*p<0.05), a more significant difference at 20 μM (**p<0.01), and an extremely significant difference at 40 μM (***p<0.001). This suggests that norathyriol can inhibit LPS- and Nigericin-induced IL-1β secretion.

[0138] Depend on Figure 3 The results showed that LPS and Nigericin stimulation significantly upregulated NLRP3 gene expression. After the addition of Norathyriol, NLRP3 gene expression was reduced compared to the LPS+Nigericin stimulation group alone, and the difference was significant at a Norathyriol concentration of 40 μM (p < 0.0001), indicating that Norathyriol can inhibit LPS and Nigericin-induced NLRP3 gene expression.

[0139] Depend on Figure 4 The results showed that LPS and Nigericin stimulation significantly upregulated IL-1β gene expression. After the addition of Norathyriol, IL-1β gene expression was significantly lower than that of the LPS+Nigericin stimulation group alone, with a highly significant difference at a Norathyriol concentration of 40 μM (p < 0.0001), indicating that Norathyriol can inhibit LPS and Nigericin-induced IL-1β gene expression.

[0140] Example 2: Ulcerative Colitis in Mice

[0141] 2.1 Animal Adaptation Feeding and Grouping

[0142] The animals used in this invention were male C57BL / 6J strain mice, 6 weeks old and weighing approximately 25g, purchased from Huafukang Experimental Animal Center. The mice were housed in an air-conditioned space with the temperature maintained at 23±1℃. Lighting was controlled with 12 hours on and 22 hours off (lights on at 08:00 and off at 20:00). Food and water were not restricted, and the mice were allowed to adapt for one week. Subsequently, the animals were randomly divided into a control group, a DSS group, a positive control group, and a Norathyriol group, with 8 mice in each group.

[0143] 2.2 Animal modeling and drug administration

[0144] Control group: Mice were given distilled water and were given 0.1 mL / kg of distilled water by gavage daily.

[0145] Model group: Mice were given free access to 2.5% DSS solution and were given 0.1 mL / kg of distilled water by gavage daily.

[0146] Positive drug group: Mice were allowed free access to 2.5% DSS solution and were administered 300 mg / kg mesalazine by gavage daily during this period.

[0147] Norathyriol group: Mice were given free access to 2.5% DSS solution and were administered 80 mg / kg Norathyriol by gavage daily. All groups were administered the medication once daily for 7 days. Successful modeling was defined as a difference in DAI and mouse body weight between the model and control groups.

[0148] 2.3 Efficacy evaluation and animal sampling

[0149] During the administration period, the weight of each mouse was measured daily, and the shape and viscosity of each mouse's feces were observed. Blood in the stool was also noted. If significant blood was observed, a photograph was taken using white paper as a substrate for recording. On the last day of administration, the mice were fasted for 16 hours but allowed free access to water. After anesthetizing the mice with tribromoethanol, the eyeballs were removed to collect blood. The colon and cecum were dissected, photographed, and their lengths measured. The colon was divided into two portions for preservation.

[0150] 2.4 Detection Indicators

[0151] (1) DAI score

[0152] During the administration period, observe the form and consistency of each mouse's feces daily, and check for any blood in the stool. Score and record the results.

[0153] The DAI scoring sheet is shown in Table 2:

[0154] Table 2 DAI Scoring Table

[0155] 0 0％ normal No rectal bleeding 1 1-5％ Slightly loose No obvious rectal bleeding 2 6-10％ semi-formed loose stool Stool color darkens 3 11-15％ mucus-like stool Blood around the anus + dark stool 4 >15％ Thin liquid stool Naked bloody stool

[0156] DAI calculation formula:

[0157] DAI score = (percentage weight loss score + stool viscosity score + bloody stool score) / 3.

[0158] (2)Weight

[0159] During the administration period, the weight of the mice was measured and recorded every morning.

[0160] (3) Inflammatory factors

[0161] After anesthesia, whole blood was collected from the eyeballs of mice, incubated overnight at 4°C, and centrifuged for 20 min (4°C, 12000 rpm / min). The supernatant serum was collected for later use. The IL-1β (STARTER, SOC3029) content in the serum of each mouse group was measured using an ELISA kit (Jianglai Biotechnology), and the results were statistically analyzed.

[0162] (4) Colon length

[0163] After euthanasia, the small colon and cecum tissues of the mice were dissected. The tissues were straightened, photographed, and their lengths were measured.

[0164] (5) Histopathological evaluation

[0165] After euthanasia, the colonic tissue obtained from dissection was divided into two portions, one of which was embedded in paraffin to prepare paraffin-embedded pathological sections. HE staining was performed on each group of pathological sections to analyze and evaluate changes in epithelial cells, inflammatory infiltration, and the number of lymph nodes in the mouse colonic pathological sections.

[0166] 2.5 Experimental Results and Analysis

[0167] (1) DAI score

[0168] DAI score results are as follows Figure 5 As shown, Norathyriol improved the DAI score in DSS-induced ulcerative colitis.

[0169] (2)Weight

[0170] Weight results as follows Figure 6 As shown, Norathyriol improves weight loss caused by DSS-induced ulcerative colitis, and its effect is superior to that of the marketed drug mesalazine.

[0171] (3) Inflammatory factors

[0172] The results are as follows Figure 7 Detection of animal serum revealed that Norathyriol can significantly reduce the elevated inflammation levels caused by ulcerative colitis and has a significant therapeutic effect on improving intestinal inflammation.

[0173] (4) Colon length

[0174] Colon length results as follows Figure 8 As shown, Norathyriol improves colonic shortening caused by DSS-induced ulcerative colitis, and its effect is superior to that of the marketed drug mesalazine.

[0175] (5) Histopathological evaluation

[0176] The results are as follows Figure 9 As shown, Norathyriol reduces inflammatory cell infiltration, epithelial shedding, and crypt structure disruption in colonic tissue.

[0177] Example 3: Inflammation induced by H1NI in mice

[0178] 3.1 Animal Adaptation Culture and Grouping

[0179] Six-week-old, 18-20g female Balb / c mice (SPF) from Beijing Vital River Laboratory Animal Technology Co., Ltd. were used. The mice were housed in individually ventilated cages at a temperature maintained at 20-24℃, with unlimited food and water. The mice were allowed to acclimatize for one week. They were then randomly divided into four groups: control group, model group, positive control group (oseltamivir phosphate 20mg / kg), and norathyriol group (80mg / kg), with 10 mice in each group.

[0180] 3.2 Animal modeling and drug administration

[0181] Mice were anesthetized using a small animal anesthesia machine, and 20 μL of 5LD virus was inoculated into the nasal cavity of the mice. Two hours after H1N1 infection, the mice were given the appropriate treatment orally.

[0182] Control group: 0.1 mL / kg of distilled water was administered by gavage daily.

[0183] Model group: 0.1 mL / kg of distilled water was administered by gavage daily.

[0184] Positive drug group: 20 mg / kg oseltamivir phosphate was administered by gavage daily.

[0185] Norathyriol group: 80 mg / kg Norathyriol was administered by gavage daily.

[0186] 3.3 Efficacy evaluation and animal sampling

[0187] Mice were administered the medication once daily for one week, with mortality and body weight monitored daily. After one week, the mice were euthanized. Lung, spleen, thymus, and peripheral blood samples were collected for further analysis.

[0188] 3.4 Experimental Results and Analysis

[0189] (1) Changes in organ weight

[0190] The results of the ratios of lung tissue, spleen tissue, and thymus tissue to body weight are as follows: Figure 10 As shown, Norathyriol improves pulmonary edema caused by pneumonia and increases indices of other organs, revealing its role in relieving lung inflammation and improving immune status.

[0191] (2)Weight

[0192] Weight results as follows Figure 11 As shown, Norathyriol improves weight loss caused by H1N1 infection.

[0193] (3) Flow cytometry analysis and results of lung tissue

[0194] The detection method is as follows:

[0195] (a) After removing the eyeballs of mice to collect blood, the lungs and spleen were removed. The spleen was weighed and placed into the corresponding well of a 24-well plate containing 1 mL of 10% FBS.

[0196] (b) Place a 200-mesh steel mesh in a cell culture dish, add 1 mL of flow cytometry buffer to the dish, grind the spleen with a volumetric flask cap while adding an appropriate amount of flow cytometry buffer, grind until only white organ membrane remains on the steel mesh.

[0197] (c) Transfer spleen cells to a 15 mL centrifuge tube and add flow cytometry staining buffer (LSHCY) to 10 mL to prepare a spleen cell suspension.

[0198] (d) Centrifuge at 300g-400g (1500rpm) for 5min and discard the supernatant.

[0199] (e) Add 2 mL of red blood cell lysis buffer, gently pipette to mix the cells, and incubate at room temperature for 4 min. (No centrifugation is required; add stop solution directly.)

[0200] (f) Add 10 mL of LSHCY to stop the lysis reaction, centrifuge at 1500 rpm for 5 min and discard the supernatant.

[0201] (g) Add 10 mL of LSHCY again to stop the lysis reaction, centrifuge at 1500 rpm for 5 min and discard the supernatant.

[0202] (h) Resuspend the cells in 1 mL of LSHCY and pipette to mix well. Count the cells in each sample.

[0203] (i) To detect CD4 and CD8 cells, take 1 mL of cell suspension and place it in a brown centrifuge tube. Set up a blank control group without antibody; set up a CD4 single-stain control group with 2 μL of APC-CD4 antibody; set up a CD8 single-stain control group with 2 μL of FITC-CD8 antibody; add 2 μL of CD4 antibody and 2 μL of CD8 antibody to each of the other test groups (the control groups here are only used for flow cytometry gating and compensation adjustment).

[0204] (10) Incubate at 4℃ in the dark for 40 minutes.

[0205] (11) After incubation, centrifuge at 300g for 5min. Discard the supernatant, wash once with PBS, resuspend the cells in 500μL PBS, pass through a 400-mesh sieve, and perform analysis.

[0206] The results are as follows Figures 12-19 Norathyriol significantly improves the imbalance of T cell differentiation caused by H1N1 infection and has a good therapeutic effect on pneumonia.

[0207] (4) Pathological changes in lung tissue

[0208] Mouse lung tissue sections were fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, and then sectioned. The sections were washed and stained with hematoxylin and eosin. Observation and imaging were performed using an inverted microscope.

[0209] Histopathological examination of mouse lung tissue was performed using HE staining to observe lesions. In the lung tissue of the model group mice, thickening of the alveolar walls, narrowing of alveolar spaces, local necrosis of alveolar epithelial cells, and significant infiltration of inflammatory cells were observed, along with extensive intravascular congestion. Norathyriol improved alveolar wall thickening and inflammatory infiltration, alleviating alveolar dilation and rupture. The positive control group showed improved alveolar wall thickening and inflammatory infiltration. Figure 20 ).

[0210] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. The use of norathyriol in the preparation of medicaments for treating inflammatory diseases, characterized in that, The structural formula of Norathyriol is as follows: ； The inflammatory disease mentioned is an inflammation caused by the influenza A virus.

2. The application according to claim 1, characterized in that, Norathyriol is the sole or primary active ingredient of the drug.

3. The application according to claim 2, characterized in that, The drug contains more than 85% Norathyriol.

4. The application according to any one of claims 1-3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients are selected from one or more combinations of wetting agents, emulsifiers, preservatives, buffers, diluents, lubricants, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

7. The application according to claim 6, characterized in that, The dosage form of the drug is injection, capsule, tablet, powder, granule, pill, microcapsule / microsphere preparation, suppository, ointment, spray, or targeted preparation.

8. The application according to claim 7, characterized in that, The administration method of the drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

9. The use of pharmaceutically acceptable salts of Norathyriol in the preparation of medicaments for treating inflammatory diseases, characterized in that, The inflammatory disease mentioned is an inflammation caused by the influenza A virus.

10. The application according to claim 9, characterized in that, The pharmaceutically acceptable salts are sodium salts, potassium salts, meglumine salts, zinc complexes, or iron complexes.

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