Application of Norathyriol in preparation of medicine for treating inflammatory diseases

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of Northyriol in preparation of a medicine for treating inflammatory diseases. According to the present invention, the Northoriol and the NLRP3 inflammasome have the binding capacity, and the Northoriol can inhibit the NLRP3 expression so as to reduce the IL-1beta gene expression at the downstream of the NLRP3 expression, such that the IL-1beta secretion in the mouse bone marrow macrophages induced by the LPS and the Nigonicin can be significantly reduced in the concentration-dependent manner; in an ulcerative colitis model, Norathyriol effectively reduces a DAI score, relieves weight loss, colon shortening and intestinal inflammation, and remarkably improves pathological injuries of colon tissues, including inflammatory cell infiltration, epithelium shedding and crypt structure destruction. Besides, the Northyriol also has a remarkable treatment effect on a pneumonia model, relieves pulmonary edema and pulmonary alveolar structure damage, improves weight loss and T cell differentiation imbalance caused by H1N1 infection, and improves organ indexes, so that the Northyriol has dual effects of relieving lung inflammation and regulating immune functions.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of Norathyriol in the preparation of drugs for treating inflammatory diseases. Background Art

[0002] Inflammatory diseases are an important research direction in the current medical field. Diseases such as ulcerative colitis, inflammation caused by influenza A virus, or pleurisy seriously threaten human health. The pathogenesis of these inflammatory diseases is complex and is mainly related to the abnormal activation of the innate immune system. Research has shown that the NLRP3 inflammasome, as an important part of the host innate immune system, plays a key role in the inflammatory response. The NLRP3 inflammasome is a multi-protein complex composed of the natural immune receptor NLRP3, the adaptor protein ASC, and the caspase-1 precursor. Its assembly can lead to the activation of caspase-1, and the activated caspase-1 subsequently promotes the cleavage of the IL-1β precursor and the IL-18 precursor, thereby generating mature and functional IL-1β and IL-18. Currently, research has shown that the abnormal activation of the NLRP3 inflammasome is related to a variety of diseases, including digestive tract inflammatory diseases (ulcerative colitis), central nervous system diseases (Parkinson's disease, Alzheimer's disease), obesity and obesity-related metabolic syndromes (type 2 diabetes), respiratory tract 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 costunolide in the preparation of drugs for preventing and treating NLRP3-mediated diseases. This patent directly covalently binds Hel to NLRP3 through a carbon-carbon double bond, interfering with the interaction between NLRP3 and NEK7, thereby inhibiting the activation of the NLRP3 inflammasome. In addition, Hel has shown significant therapeutic effects in several mouse models of NLRP3-mediated diseases.

[0004] CN116473966A discloses the application of SB-222200 in inhibiting the activation of the NLRP3 inflammasome. This patent directly binds to the NLRP3 protein, inhibits the assembly of the NLRP3 inflammasome, also inhibits the oligomerization and speck formation of the ASC protein during the activation of the NLRP3 inflammasome, inhibits the cleavage of GSDMD and the resulting pyroptosis, inhibits the oligomerization of the NLRP3 protein itself and the interaction between the NLRP3 protein and the ASC protein. At the same time, this SB-222200 can also relieve peritonitis and inflammatory bowel disease.

[0005] Patent CN113288936A discloses a pharmaceutical composition for treating ulcerative colitis and its application. The composition includes Stephania extract. This patent shows the therapeutic effects of this pharmaceutical composition in aspects such as alleviating weight loss in DSS-induced ulcerative colitis mice, reducing the DAI score, increasing colon length, improving the gross morphology score of the colon, alleviating the degree of inflammatory infiltration, and inhibiting the accumulation of MPO enzyme in colon tissues. However, this patent still has the problem of further screening and optimizing the preparation methods of Stephania extract and Gardenia jasminoides Ellis extract to improve the therapeutic effect and bioavailability of the pharmaceutical composition.

[0006] Patent CN116920012A relates to the application of Clinopodium polycephalum in the preparation of drugs for treating ulcerative colitis. This patent proves that the Clinopodium polycephalum extract can effectively reduce the DAI score, inhibit the levels of inflammatory factors in plasma, alleviate the inflammatory response of colon mucosal tissues, and play a protective role on colon mucosa. However, this patent still has the problem of further studying the optimal dosage and administration method of Clinopodium polycephalum extract in the treatment of ulcerative colitis to improve the therapeutic effect and safety of the drug.

[0007] Norathyriol is the deglycosylated product of mangiferin and belongs to flavonone compounds, having various biological activities such as antioxidant, anti-inflammatory, and anti-tumor. Its structural characteristics make it more lipophilic than mangiferin, which may affect its absorption and mechanism of action in vivo. As a natural compound with potential medicinal value, Norathyriol has certain development prospects in functional food 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 NLRP3 inflammasome such as ulcerative colitis, inflammation caused by influenza A virus, or pleurisy, the application value of Norathyriol needs to be further explored. Summary of the Invention

[0009] In view of the above deficiencies, the present invention first proposes the unique curative effect of Norathyriol in the treatment of inflammatory diseases. Experimental studies show that Norathyriol exerts its therapeutic effects through multiple targets and multiple pathways. The present invention provides a new option for the clinical treatment of inflammatory diseases and has important clinical application value.

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

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

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

[0013]

[0014] Specifically, the inflammatory disease is a disease 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 H1NI virus.

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

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

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

[0024] Specifically, the drug further comprises pharmaceutically acceptable excipients.

[0025] Preferably, the pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0026] Preferably, 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, methyl paraben, propyl paraben, magnesium stearate, and mineral oil.

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

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

[0029] Preferably, the administration route of the drug can be oral administration.

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

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

[0032] In another aspect, the present invention provides the use of an isomer of Norathyriol in the preparation of a drug for treating inflammatory diseases.

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

[0034] Specifically, the inflammatory disease is a disease 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 H1NI virus.

[0042] Specifically, the drug further comprises a pharmaceutically acceptable excipient.

[0043] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, solubilizers, thickeners, stabilizers, sweeteners and fragrances.

[0044] Preferably, 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.

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

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

[0047] Preferably, the administration route of the drug can be oral administration.

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

[0049] Specifically, the solvate includes but is not limited to: hydrate, methanolate, ethanolate, isopropanolate or DMSO solvate.

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

[0051] Preferably, the inflammatory diseases are selected from:

[0052] (a) Digestive tract inflammatory diseases;

[0053] (b) Respiratory tract inflammatory diseases;

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

[0055] Preferably, the digestive tract inflammatory disease is ulcerative colitis; the respiratory tract inflammatory disease 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 H1NI virus.

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

[0059] Preferably, the pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifying agents, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0060] Preferably, 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, methyl paraben, propyl paraben, 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 route of the drug is selected from oral administration, intravenous injection, topical, intradermal, or subcutaneous injection.

[0063] Preferably, the administration route of the drug can be oral administration.

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

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

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

[0067] Preferably, the inflammatory diseases are 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 H1NI virus.

[0074] Specifically, the drug further comprises a pharmaceutically acceptable excipient.

[0075] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an excipient, a diluent, a lubricant, an antibacterial agent, a suspending agent, a suspending aid, a solubilizer, a thickening agent, a stabilizer, a sweetening agent, and a flavoring agent.

[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, a capsule, a tablet, a powder, a granule, a pill, a microcapsule microsphere preparation, a suppository, an ointment, a spray, or a targeted preparation.

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

[0079] Preferably, the administration route of the drug may be oral administration.

[0080] On the other hand, the present invention provides the use of the co-crystal of Norathyriol in the preparation of a drug for treating inflammatory diseases.

[0081] Specifically, the co-crystal former of the co-crystal may 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 a disease mediated by abnormal activation of the NLRP3 inflammasome.

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

[0084] (a) Digestive tract inflammatory diseases;

[0085] (b) Respiratory tract inflammatory diseases;

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

[0087] Preferably, the digestive tract inflammatory disease is ulcerative colitis; the respiratory tract inflammatory disease 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 may be an H1N1 virus.

[0090] Specifically, the drug further comprises a pharmaceutically acceptable excipient.

[0091] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a suspending aid, a solubilizer, a thickening agent, a stabilizer, a sweetening agent, and a flavoring agent.

[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, a capsule, a tablet, a powder, a granule, a pill, a microcapsule and microsphere preparation, a suppository, an ointment, a spray, or a targeted preparation.

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

[0095] Preferably, the administration route of the drug may be oral administration.

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

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

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

[0099] (3) Norathyriol improves pulmonary edema caused by pneumonia and increases the indices of other organs, revealing its role in alleviating lung inflammation and improving the immune state; Norathyriol improves the 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 the thickening of the alveolar wall and inflammatory infiltration and alleviates alveolar dilation and rupture. Description of the Drawings

[0100] Figure 1 It is the SPR sensing diagram analysis diagram in Example 1.

[0101] Figure 2 It is for detecting the content of IL-1β in the supernatant of ELISA in Example 1. The "*" in the figure represents p < 0.05 compared with the group stimulated only with LPS + Nigericin; "**" represents p < 0.01 compared with the group stimulated only with LPS + Nigericin; "***" represents p < 0.001 compared with the group stimulated only with LPS + Nigericin.

[0102] Figure 3 It is for detecting the relative expression level of NLRP3 gene by real-time fluorescence quantitative method in Example 1. The "***" in the figure represents p < 0.001 compared with the group stimulated only with LPS + Nigericin.

[0103] Figure 4 It is for detecting the relative expression level of IL-1β gene by real-time fluorescence quantitative method in Example 1. The "****" in the figure represents p < 0.0001 compared with the group stimulated only with LPS + Nigericin.

[0104] Figure 5 It is the DAI score result diagram of each group in Example 2.

[0105] Figure 6 It is the weight change trend diagram of each group in Example 2.

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

[0107] Figure 8 It is the colon length result diagram of each group in Example 2.

[0108] Figure 9 It is the histopathology diagram of each group in Example 2.

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

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

[0111] Figure 12 This is the CD4+ T cell flow cytometry graph of the lung tissue blank group in Example 3.

[0112] Figure 13 This is the flow cytometry graph of CD4+ T cells in the lung tissue model group in Example 3.

[0113] Figure 14 This is a flow cytometry graph of CD4+ T cells in the lung tissue-positive drug group in Example 3.

[0114] Figure 15 This is the flow cytometry graph of CD4+ T cells in the lung tissue Norathyriol group in Example 3.

[0115] Figure 16 This is the CD8+ T cell flow cytometry graph of the lung tissue blank group in Example 3.

[0116] Figure 17 This is the flow cytometry graph of CD8+ T cells in the lung tissue model group in Example 3.

[0117] Figure 18 This is a flow cytometry graph of CD8+ T cells in the lung tissue-positive drug group in Example 3.

[0118] Figure 19 This is the flow cytometry graph of CD8+ T cells in the lung tissue Norathyriol group in Example 3.

[0119] Figure 20 This is the lung tissue pathology diagram in Example 3. DETAILED DESCRIPTION

[0120] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

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

[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 method

[0126] In the experiment, a CM5 chip was used. The recombinant purified NLRP3 protein was immobilized on the chip surface by the EDC / NHS chemical coupling method. The coupling buffer was 10 mM sodium acetate buffer at pH 5.5. After immobilization, Norathyriol solutions with different concentrations (such as 0.156 - 10 μM) were sequentially injected into the chip. The solvent was HBS-EP+ buffer containing 2% DMSO. The injection flow rate was 30 μL / min, the binding time was 120 seconds, and the dissociation time was 300 seconds. The experimental data was processed by Biacore 8K Evaluation software. After subtracting the reference signal of the blank channel, a 1:1 Langmuir model was used for fitting to calculate the binding ability of Norathyriol to NLRP3 protein.

[0127] (2) Results

[0128] The results are shown in Table 1:

[0129] Table 1

[0130] Quality Affinity ChP(RU) Steady state affinity KD(M) Rmax(RU) Offset(RU) <![CDATA[1.94×10 2 > <![CDATA[4.10×10 -5 > 186.1 7.5

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

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

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

[0134] (1) Experimental method:

[0135] Differentiate mouse bone marrow macrophages BMDMs in RPMI-1640 medium supplemented with 20 ng / mL M-CSF for 7 days. Seed 12-well plates at 1×10 5 cells / well. After adherent for 24 h, replace with fresh medium containing 50 ng / mL LPS and the specified drug concentration (Norathyriol concentrations are 0 μM, 5 μM, 10 μM, 20 μM, 40 μM respectively) and culture for 3 h. Then add 10 μM Nigericin and stimulate for 30 min. Subsequently, use an ELISA kit (STARTER, S0C3029) to measure the content of IL-1β in the supernatant. Use the Trizol method to extract total cellular RNA, and use real-time fluorescence quantitative method to measure the expression levels of NLRP3 and IL-1β genes in cells.

[0136] (2) Experimental results

[0137] From Figure 2 the results, it can be seen that after adding Norathyriol, the secretion of IL-1β shows a concentration-dependent decrease. Statistical symbols show that there is a significant difference when the Norathyriol concentration is 10 μM (*p < 0.05), the difference is more significant at 20 μM (**p < 0.01), and the difference is extremely significant at 40 μM (***p < 0.001). This indicates that Norathyriol can inhibit the secretion of IL-1β induced by LPS and Nigericin.

[0138] From Figure 3 the results, it can be seen that LPS and Nigericin stimulation significantly up-regulates the expression of NLRP3 gene. After adding Norathyriol, the expression of NLRP3 gene is decreased compared with the group only stimulated by LPS + Nigericin, and the difference is significant when the Norathyriol concentration is 40 μM (****p < 0.0001), indicating that Norathyriol can inhibit the expression of NLRP3 gene induced by LPS and Nigericin.

[0139] From Figure 4 the results, it can be seen that LPS and Nigericin stimulation significantly up-regulates the expression of IL-1β gene. After adding Norathyriol, the expression of IL-1β gene is significantly decreased compared with the group only stimulated by LPS + Nigericin, and the difference is extremely significant when the Norathyriol concentration is 40 μM (****p < 0.0001), indicating that Norathyriol can inhibit the expression of IL-1β gene induced by LPS and Nigericin.

[0140] Example 2 Mouse ulcerative colitis

[0141] 2.1 Animal Adaptive Feeding and Grouping

[0142] The animals used in this invention are male C57BL / 6J strain mice purchased from Huafukang Experimental Animal Center, 6 weeks old and weighing about 25 g. The mice are raised in an air-conditioned space with the temperature maintained at 23 ± 1 °C, and the lighting is controlled with 12 hours of light and 12 hours of darkness (lights on at 08:00 and lights off at 20:00). Food and water are not restricted, and the mice are adaptively fed for one week. Subsequently, the animals are randomly divided into a control group, a DSS group, a positive drug group, and a Norathyriol group, with 8 mice in each group.

[0143] 2.2 Animal Model Establishment and Drug Administration

[0144] Blank group: The mice drink distilled water and are intragastrically administered 0.1 mL / kg of distilled water every day during the period.

[0145] Model group: The mice freely drink 2.5% DSS solution and are intragastrically administered 0.1 mL / kg of distilled water every day during the period.

[0146] Positive drug group: The mice freely drink 2.5% DSS solution and are intragastrically administered 300 mg / kg of mesalazine every day during the period.

[0147] Norathyriol group: The mice freely drink 2.5% DSS solution and are intragastrically administered 80 mg / kg of Norathyriol every day during the period. Each group above is administered the drug once a day, and the experimental period is 7 days in total. If there are differences in DAI and mouse body weight between the model group and the control group, it is considered that the model establishment is successful.

[0148] 2.3 Efficacy Evaluation and Animal Sampling

[0149] During the drug administration period, the body weight of each mouse is weighed every day, the fecal morphology and viscosity of each mouse are observed, and whether there is bloody stool is observed. If the bloody stool of the mouse is obvious, it can be photographed and recorded with white paper as the background. On the last day of animal drug administration, the mice are fasted but not water-deprived for 16 h. After anesthetizing the mice with tribromoethanol, blood is taken by eye socket enucleation. The colon and cecum of the mice are dissected, photographed, and the length is measured. The colon is divided into 2 parts for preservation.

[0150] 2.4 Detection Indexes

[0151] (1) DAI Score

[0152] During the drug administration period, the fecal morphology, viscosity, and whether there is bloody stool of each mouse are observed every day, scored, and recorded.

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

[0154] Table 2 DAI Score Scoring Table

[0155] Score Percentage of weight loss Stool consistency Blood in stool 0 0％ Normal No blood in stool 1 1-5％ Slightly loose No obvious blood in stool 2 6-10％ Semi-formed loose stool Darkening of stool color 3 11-15％ Mucus-like stool Blood around anus + dark stool 4 >15％ Watery stool Gross hematochzia

[0156] DAI calculation formula:

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

[0158] (2) Body weight

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

[0160] (3) Inflammatory factors

[0161] After anesthesia, the eyeballs of the mice were removed to collect whole blood, which was left standing overnight at 4°C and then centrifuged for 20 min (4°C, 12,000 rpm / min). The upper serum was taken for standby. The content of IL-1β (STARTER, S0C3029) in the sera of each group of mice was measured using an ELISA kit (Jianglai Biotech), and the results were statistically analyzed.

[0162] (4) Colon length

[0163] After the mice were euthanized, the small intestine, colon, and cecum tissues were dissected. They were straightened, measured, photographed, and the length was measured.

[0164] (5) Histopathological evaluation

[0165] After the mice were euthanized, the dissected colon tissues were divided into two parts. One part was embedded in paraffin to prepare paraffin pathological sections. HE staining was performed on the pathological sections of each group, and the changes in epithelial cells, inflammatory infiltration, and the number of lymph nodes in the pathological sections of the mouse colon were analyzed and evaluated.

[0166] 2.5 Experimental results and analysis

[0167] (1) DAI score

[0168] The DAI score results are as Figure 5 shown. Norathyriol improved the DAI score of DSS-induced ulcerative colitis.

[0169] (2) Body weight

[0170] The body weight results are as Figure 6 shown. Norathyriol improved the weight loss caused by DSS-induced ulcerative colitis, and its effect was better than that of the marketed drug mesalazine.

[0171] (3) Inflammatory factors

[0172] The results are as Figure 7 shown. By detecting the sera of the animals, it was found that Norathyriol could significantly reduce the elevated inflammatory level caused by ulcerative colitis and had an obvious therapeutic effect on improving intestinal inflammation.

[0173] (4) Colon length

[0174] The results of colon length are as Figure 8 shown. Norathyriol improved the colon shortening caused by DSS-induced ulcerative colitis, and its effect was better than that of the marketed drug mesalazine.

[0175] (5) Histopathological evaluation

[0176] The results are as Figure 9 shown. Norathyriol alleviated the infiltration of inflammatory cells, epithelial shedding and crypt structure damage in the colon tissue.

[0177] Example 3 Inflammation induced by H1NI in mice

[0178] 3.1 Animal adaptation culture and grouping

[0179] Specific pathogen-free (SPF) female Balb / c mice, 6 weeks old, weighing 18 - 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in individually ventilated cages at a temperature of 20 - 24 °C with unlimited food and water, and the mice were allowed to adapt to the environment for one week. The mice were randomly divided into 4 groups: control group, model group, positive drug group (oseltamivir phosphate 20 mg / kg), and Norathyriol group (80 mg / kg), with 10 mice in each group.

[0180] 3.2 Animal model establishment and drug administration

[0181] The mice were anesthetized using a small animal anesthesia machine, and 20 μL of 5LD virus was inoculated into the nasal cavity of the mice. After H1N1 infection, 2 hours later, the mice were orally administered the corresponding treatment.

[0182] Control group: gavaged with 0.1 mL / kg distilled water every day.

[0183] Model group: gavaged with 0.1 mL / kg distilled water every day.

[0184] Positive drug group: gavaged with 20 mg / kg oseltamivir phosphate every day.

[0185] Norathyriol group: gavaged with 80 mg / kg Norathyriol every day.

[0186] 3.3 Efficacy evaluation and animal sampling

[0187] The drug was administered once a day for a one-week treatment period, and the mortality and body weight were 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 ratio of lung tissue, spleen tissue and thymus tissue to body weight is as follows Figure 10 As shown, Norathyriol improves pulmonary edema caused by pneumonia and increases the indexes of other organs, revealing that it has the effect of alleviating lung inflammation and improving immune status.

[0191] (2)Weight

[0192] Weight results as Figure 11 As shown, Norathyriol ameliorates H1N1 infection-induced weight loss.

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

[0194] The detection method is as follows:

[0195] (a) After the mouse's eyeballs were removed and blood was collected, the lungs and spleen were removed. The spleen was weighed and placed in 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 the cap of a volumetric flask while adding appropriate flow cytometry buffer until only white organ membranes remain on the steel mesh.

[0197] (c) Transfer the 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 5 min and discard the supernatant.

[0199] (e) Add 2 mL of red blood cell lysis buffer and gently pipette to mix the cells. Incubate at room temperature for 4 min. (Add stop buffer directly without centrifugation).

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

[0201] (g) Add 10 mL of LSHCY again to terminate 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 evenly. Count the cells of each sample.

[0203] (i) Take the cell suspension to detect CD4 and CD8 cells. Take 1 mL of the cell suspension and place it in a brown centrifuge tube. Set up a blank control group without adding antibodies; set up a CD4 single-staining control group and add 2 μL of APC-CD4 antibody; set up a CD8 single-staining control group and add 2 μL of FITC-CD8 antibody; add 2 μL of CD4 antibody and 2 μL of CD8 antibody to each of the remaining test groups (the control groups here are only used for gating and compensating adjustment of the flow cytometer).

[0204] (10) Incubate at 4 °C in the dark for 40 min.

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

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

[0207] (4) Histopathological changes in the lung tissue

[0208] Fix the lung tissue sections of mice in 4% paraformaldehyde for 48 hours, embed them in paraffin and section. Wash the sections and stain them with hematoxylin and eosin. Observe and image using an inverted microscope.

[0209] Perform histopathological examination on the lung tissue of mice with HE staining to observe the lesions. In the lung tissue of the model group mice, thickening of the alveolar wall, narrowing of the alveolar space, local necrosis of alveolar epithelial cells, obvious infiltration of inflammatory cells were observed, and extensive congestion was observed in the blood vessels. Norathyriol improved the thickening of the alveolar wall and inflammatory infiltration, and alleviated alveolar dilation and rupture. The positive drug group showed improvement in the thickening of the alveolar wall and inflammatory infiltration ( Figure 20 ).

[0210] The above detailed description is a specific description of one of the feasible embodiments 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 changes 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 this invention patent shall be subject to the appended claims.

Claims

1. Use of norathyriol in the preparation of a medicament for treating inflammatory diseases, characterized in that, The structural formula of Norathyriol is as follows:

2. The application according to claim 1, characterized in that, The inflammatory disease is a disease mediated by abnormal activation of NLRP3 inflammasome.

3. The application according to claim 1, characterized in that, The inflammatory disease is selected from: (a) Inflammatory diseases of the digestive tract; (b) Inflammatory diseases of the respiratory tract.

4. The application according to claim 3, wherein 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.

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

6. The application according to claim 5, wherein The content of Norathyriol in the drug is greater than 85%.

7. The application according to any one of claims 1-6, characterized in that, The drug further comprises pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients are selected from one or a combination of two or more of wetting agents, emulsifying agents, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and fragrances.

9. The application according to claim 8, wherein, 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.

10. The application according to claim 9, wherein The dosage form of the drug is injection, capsule, tablet, powder, granule, pill, microcapsule microsphere preparation, suppository, ointment, spray or targeted preparation.

11. The application according to claim 9, characterized in that, The administration mode of the drug is selected from oral administration, intravenous injection, topical, intradermal or subcutaneous injection.

12. Use of an isomer of Norathyriol in the preparation of a drug for treating inflammatory diseases.

13. The application according to claim 12, characterized in that, The isomer is a positional isomer.

14. Use of a solvate of Norathyriol in the preparation of a drug for treating inflammatory diseases.

15. The application according to claim 14, characterized in that, The solvate is a hydrate, methanolate, ethanolate, isopropanolate or DMSO solvate.

16. Use of a pharmaceutically acceptable salt of Norathyriol in the preparation of a drug for treating inflammatory diseases.

17. The application according to claim 16, characterized in that, The pharmaceutically acceptable salt is sodium salt, potassium salt, meglumine salt, zinc complex or iron complex.

18. Use of a cocrystal of Norathyriol in the preparation of a drug for treating inflammatory diseases.

19. The application according to claim 18, characterized in that, The cocrystal former of the cocrystal is selected from one or more of oxalic acid, citric acid, succinic acid, nicotinamide, urea, caffeine, isonicotinamide, glucose or lactose.

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