Pharmaceutical composition for treating pulmonary inflammatory disease, preparation thereof, and use thereof

Abstract

Provided are a pharmaceutical composition for treating a pulmonary inflammatory disease, a preparation thereof, and use thereof. The pharmaceutical composition comprises the following components: 1-100 parts of a thesine component and 1-20 parts of a matrine component, wherein the thesine component is selected from one or a combination of thesine I, thesine II, thesine III, thesine A, and thesine B, and the matrine component is selected from one or a combination of matrine, sophocarpine, thesium alkaloid A, and thesium alkaloid B. The thesine component reduces toxicity of the matrine component at high concentrations, can inhibit hepatorenal toxicity caused by the matrine component, and can inhibit ROS generation caused by the matrine component.

Description

A pharmaceutical composition for treating pulmonary inflammatory diseases, its preparation and application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 28, 2023, with application number 2023118374770 and invention name “A pharmaceutical composition for treating inflammatory diseases of the lungs, its preparation and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the technical field of pneumonia pharmaceutical compositions, and in particular to a pharmaceutical composition for treating inflammatory lung diseases, and its preparation and application. Background Art

[0003] Inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD) and pneumonia, are highly prevalent and devastating, with their pathogenesis closely linked to inflammatory responses. For example, COPD has a high incidence and mortality rate worldwide, and the elderly are a high-risk group for COPD. COPD is a preventable and treatable disease characterized by persistent airflow limitation, associated with chronic inflammation and autoimmune responses caused by pathogenic microorganisms, cigarette smoke, and toxic particles or gases. Oxidative stress induced by harmful substances stimulates alveolar macrophages to secrete various cytokines, such as interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α), which promote the release of proteases, increase epithelial permeability, and increase lipid peroxide levels. These factors lead to the retention and activation of neutrophils in the airways and lungs, significantly increasing the release of inflammatory factors, further exacerbating the inflammatory response in the airways, lungs, and systemic airway. Respiratory diseases are closely linked to inflammatory responses, and developing therapeutic drugs to treat respiratory diseases is an effective strategy to address inflammation.

[0004] Acute or chronic inflammatory diseases result in the production of large amounts of nitric oxide (NO). Sustained high levels of NO in the human body can directly damage DNA and proteins, leading to tissue damage. Therefore, NO production inhibition activity can reflect the anti-inflammatory activity of a compound. Quinone reductase (QR) is a Phase II detoxification enzyme that catalyzes the reduction of quinones, eliminating reactive oxygen species (ROS) in the body. Phase II detoxification enzymes are generally believed to be primarily responsible for the metabolic detoxification of harmful oxidants and chemical carcinogens. Therefore, QR-inducing activity can reflect a compound's ability to inhibit inflammation and oxidative stress, as well as its preventive effect on inflammatory-related diseases such as COPD and pneumonia. Research has demonstrated that oxidative stress and inflammation can interact, with oxidative stress inducing inflammation and inflammation exacerbating oxidative stress. Persistent oxidative stress and inflammation can lead to the release of massive amounts of inflammatory mediators, altered cell permeability, and necrosis. Furthermore, elevated ROS levels can damage biomacromolecules such as DNA, proteins, and lipids, potentially contributing to a range of inflammatory diseases.

[0005] The formation of the NLRP3 inflammasome activates procaspase-1, which in turn generates caspase-1, promoting the maturation and secretion of the inflammatory cytokine IL-1β. Many serious human diseases, such as COPD, asthma, allergic airway inflammation, pneumonia, diabetic nephropathy, gout, non-alcoholic steatohepatitis, myocardial infarction, hypertension, and Alzheimer's disease, are closely associated with excessive activation of the NLRP3 inflammasome. Therefore, the development of drugs that inhibit NLRP3 inflammasome activation is of great significance.

[0006] Ferroptosis is a unique form of programmed cell death that is closely associated with the development and progression of many lung diseases, including acute lung injury, chronic obstructive pulmonary disease, asthma, cystic fibrosis, lung infections (bacterial, viral, and fungal), and vascular lung diseases (ischemia-reperfusion injury and pulmonary hypertension). Ferroptosis is primarily characterized by intracellular iron accumulation, glutathione depletion, inactivation of glutathione peroxidase, and promotion of lipid peroxidation. Ferroptosis can be manipulated by multiple signaling pathways, including p62 kelch-like ECH-associated protein (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), solute carrier family 7, member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and acyl-CoA synthetase long-chain family member 4 (ACSL4). Numerous interventions, including ferroptosis inducers and inhibitors, iron chelators, lipid peroxidation inhibitors, and antioxidants, have been introduced for ferroptosis-related lung diseases.

[0007] T cell-mediated pulmonary inflammatory response has been shown to play a key role in lung diseases. CD4 cells differentiate into mature CD4 cells, which then differentiate into different cell subtypes, including Th1, Th2, Th17, and regulatory Treg cells. Different cell subpopulations secrete distinct cytokines and chemokines, which mediate (or inhibit) the recruitment of inflammatory cells to the site of infection. This promotes (or inhibits) the progression of chronic lung diseases such as chronic obstructive pulmonary disease (COPD), lung cancer, cystic fibrosis, and asthma. There is a close crosstalk between lung immunity and the intestinal microbiota, mediated by the lymphatic and effusion circulation systems. In COPD, exogenous particulate matter or cigarette smoke can cause intestinal inflammatory responses and alterations in the composition of the intestinal microbiota. Furthermore, derived components of the intestinal microbiota or their metabolites can damage the intestinal barrier, leading to local and systemic immune responses, which in turn affect the immune response and pathological damage in the lungs. The lung-gut interaction is bidirectional. The lungs can also influence the intestine through mechanisms such as lymphocyte migration and inflammatory cytokines.

[0008] Thesium chinense Turcz. is a plant of the genus Thesium in the family Santalaceae. The whole plant is used medicinally. It has a pungent, slightly bitter flavor and a cold nature, and enters the spleen, kidney, and lung meridians. It has the effects of clearing heat and detoxifying, tonifying the kidneys, and astringing semen. Research on the chemical components of Thesium chinense Turcz. is relatively limited, with over 60 compounds reported in the literature, including flavonoids, alkaloids, organic acids, and terpenes. The material basis and mechanism of action of Thesium chinense Turcz. in treating inflammatory diseases remain unclear.

[0009] The main lycoside components include lycoside I, lycoside II, and lycoside III. According to literature reports, lycoside I has anti-inflammatory and anti-diabetic activities. lycoside II has anti-inflammatory, antioxidant, neuroprotective, anti-osteoporosis, and anti-cancer effects. lycoside III has anti-inflammatory, antioxidant, osteoporosis treatment, and anti-cancer effects. Thus, lycoside components have broad application prospects in the field of new drug research and development. lycoside I, lycoside II, and lycoside III are the main components of lycoside, but purification is difficult, and there are currently no reports of large-scale preparations of pure lycoside I, lycoside II, and lycoside III from lycoside. Furthermore, lycoside components have poor water solubility and low bioavailability. Previous literature only discovered three alkaloids from lycoside, but the content was extremely low, among which sophocarpine is a matrine component.

[0010] Matrine components belong to a type of quinolizidine alkaloids, and their molecular skeleton is composed of two quinolizidine rings fused together. This type of alkaloids is widely found in the leguminous plants Sophora flavescens, Sophora alopecuroides, and Sophora flavescens roots. Representative compounds include matrine, sophoridine, sophorocarpine, sophoramine, and sophorolidine. Matrine has multiple pharmacological activities, such as anti-tumor, anti-arrhythmic, anti-diabetic, immunosuppressive, anti-inflammatory, antibacterial, antiviral, antiparasitic, anti-fibrotic, and neuroprotective. Matrine alkaloids are used in clinical practice due to their wide range of pharmacological activities. However, their non-negligible toxicity limits their development as therapeutic drugs, especially their long-term use can cause liver and kidney damage.

[0011] Application Contents

[0012] The purpose of the embodiments of the present application is to provide a pharmaceutical composition for treating inflammatory lung diseases, its preparation and application, so as to solve the technical problem that there is no drug in the prior art that can effectively treat inflammatory lung diseases. Technical Solutions

[0013] The technical solution adopted in the embodiment of this application is:

[0014] In a first aspect, the present application provides a pharmaceutical composition for treating pulmonary inflammatory diseases, comprising the following components:

[0015] 1-100 parts of celery scutellariae components,

[0016] 1-20 parts of matrine components;

[0017] The general structural formula of the celery extract component is:

[0018] wherein R4 is H or a group; R1, R2, R3, and R5 are independently selected from one of the following groups: H, CH3, CH3CO, glucosyl, rhamnosyl, glucose rhamnosyl, glucose glucosyl, xylosyl, and sucrose;

[0019] The general structural formula of the matrine component is:

[0020] Wherein, R6, R7, and R8 are independently selected from one of the following groups: H, OH, OCH3, and CH3COO.

[0021] In some embodiments, the pharmaceutical composition comprises the following components:

[0022] 60-66 parts of the celeriac component,

[0023] The matrine component is 2-5 parts.

[0024] In some embodiments, the lycoside component is selected from at least one of lycoside I, lycoside II, lycoside III, lycoside A, and lycoside B;

[0025] In some embodiments, the matrine component is selected from at least one of matrine, sophoracarpine, lycopodipine A, and lycopodipine B.

[0026] In some embodiments, the pharmaceutical composition comprises the following components:

[0027] In some embodiments, the pharmaceutical composition comprises the following components:

[0028] 10-15 parts of the described celery scutellarin III,

[0029] 1 part of described matrine.

[0030] In some embodiments, the method for extracting celastrol A and celastrol B comprises the following steps:

[0031] The dried whole herb of Herba Lycoris Radiatae is crushed, soaked at 20-40°C for 1-10 hours, and then heated and refluxed with 60-80% ethanol to obtain an extract; the extract is filtered and concentrated in sequence to obtain a total extract;

[0032] After the total extract was suspended in water, it was extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain various organic extracts;

[0033] The ethyl acetate extract is separated by normal phase silica gel column chromatography, wherein first, gradient elution is performed using an eluent of petroleum ether and ethyl acetate in a volume ratio of 0.1-1:0.1-1, and then gradient elution is performed using an eluent of ethyl acetate / methanol in a volume ratio of 0.1-1:0.1-1; 8 fractions are collected in chronological order to obtain first elution products E1-E8;

[0034] After the first elution product E7 fraction was passed through an MCI column to remove the pigment, gradient elution was performed using a methanol / water eluent with a volume ratio of 1-3:0.1-7, and 8 fractions were collected in chronological order to obtain the second elution products E7a-h;

[0035] The second eluted product E7d was separated by a normal silica gel column using a gradient elution of dichloromethane / methanol in a volume ratio of 0.1 to 30:1. Eight fractions were collected in chronological order to obtain the third eluted products E7a-h.

[0036] The third elution product E7d5 was separated and purified by Sephadex LH-20 and C18 reverse phase silica gel to obtain compounds celastrol A and celastrol B.

[0037] Wherein, the structural formula of the celastrol A is as follows:

[0038] The structural formula of the celery scutellarin B is as follows:

[0039] In some embodiments, the extraction method of the lycopodium alpinum A and the lycopodium alpinum B is:

[0040] The whole herb of Herba Lycopodii is crushed, extracted with a 0.1-0.5 mol / L hydrochloric acid aqueous solution, the extract is adjusted to alkaline, and then repeatedly extracted with dichloromethane, and concentrated under reduced pressure to obtain a total alkaloid extract;

[0041] The total alkaloid extract was separated by a normal phase silica gel column and eluted with a petroleum ether / dichloromethane / ammonia water eluent in a volume ratio of 1:1:0.02-0.03; then gradient elution was performed with a dichloromethane / methanol eluent in a volume ratio of 6-97:3-4, and 10 fractions were collected in chronological order to obtain the fourth elution products S1-S10;

[0042] The fourth eluted product S5 was purified by Sephadex LH-20 column chromatography using methanol as eluent, and the purified S5 was separated by HPLC using a mobile phase having a pH of 3 to 4 and a methanol content of 5% to 10% by volume;

[0043] Impurities were removed by macroporous adsorption resin column to obtain lycopodium B and lycopodium A;

[0044] Wherein, the structural formula of the cyperine A is as follows:

[0045] The structural formula of the lycopodipine B is as follows:

[0046] In some embodiments, the extraction process of the lycopodiola rosea I, the lycopodiola rosea II, and the lycopodiola rosea III comprises the following steps:

[0047] The dried whole herb of Herba Lycopodii is crushed and soaked at 20-40°C for 1-10 hours, and then subjected to heating and reflux extraction with a 60-80% by weight ethanol aqueous solution; the extract is filtered and concentrated to obtain a total extract;

[0048] After the total extract was suspended in water, it was extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain various organic extracts;

[0049] The ethyl acetate extract was added with water and passed through an MCI column, and then eluted with methanol-water solutions with a mass percentage of 20%-30%, 40%-50%, and 60%-70% in sequence to obtain the first fraction, the second fraction, and the third fraction in sequence;

[0050] The first fraction is then eluted with pure water, ethanol-water solutions with a mass percentage of 20%-30%, 40%-50%, 60%-70%, and pure ethanol, to obtain component a, component b, component c, component d, and component e, respectively;

[0051] wherein the component b is purified by gel chromatography using methanol as the eluent and recrystallized to obtain celastrol I;

[0052] The component C is purified by gel chromatography using methanol as eluent, and recrystallized to obtain celastrol II;

[0053] The third fraction is purified by gel chromatography using methanol as eluent and recrystallized to obtain celastrol III.

[0054] In a second aspect, the present application provides a preparation for inflammatory diseases of the lungs, wherein the mass percentage of the above-mentioned pharmaceutical composition as an active ingredient in the preparation is 0.1%-99.9%, and the rest is pharmaceutically acceptable excipients; wherein the dosage form of the preparation is tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, lozenges, granules, electuary, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, drops or patches.

[0055] In a third aspect, the present application provides the use of the above-mentioned pharmaceutical composition for treating inflammatory lung diseases in the preparation of drugs for chronic obstructive pulmonary disease, acute lung injury or lower respiratory tract infection. Beneficial effects

[0056] The first beneficial effect provided by the embodiments of the present application is that: the pharmaceutical composition provided by the present application mainly includes celastrol components and matrine components. The celastrol components have QR inducing activity, can inhibit LPS-induced excessive production of NO in RAW264.7 mouse macrophages, reduce LPS-induced pathological changes in mouse lung tissue, inhibit the increase of inflammatory factor IL-1β in the alveolar lavage fluid of mice induced by LPS, and can reduce the number of inflammatory cells in the blood of mice, thereby exerting a lung protective effect and alleviating lung inflammation. The synergistic and enhancing effects of chelidonin components and matrine components, the two of which play a role in treating lung diseases through different mechanisms of action. Among them, chelidonin components have a new mechanism for treating acute lung injury by regulating ferroptosis-related pathway proteins SLC7A11 and GPX4. At the same time, matrine components can inhibit the excessive activation of NLRP3 inflammasome and inhibit NF-kB activation; the two synergistically exert lung protection effects through different mechanisms of action; and the binding of chelidonin and matrine components to key inflammatory regulatory proteins has better anti-inflammatory effects. Therefore, the two components have an interaction of "enhancing efficacy and reducing toxicity, complementary mechanisms, and mutual solubility", which is conducive to wide application.

[0057] The second beneficial effect provided by the embodiments of the present application is that: a preparation for inflammatory lung diseases is provided, wherein the mass percentage of the above-mentioned pharmaceutical composition as an effective component in the preparation is 0.1%-99.9%; therefore, the provided preparation has the effect of alleviating lung inflammation, protecting the lungs, has excellent anti-inflammatory effects, and is conducive to wide application.

[0058] The third beneficial effect provided by the embodiments of the present application is that the pharmaceutical composition for treating inflammatory lung diseases has excellent anti-inflammatory effects, can effectively reduce lung inflammation, and can be widely used in the preparation of drugs for chronic obstructive pulmonary disease, acute lung injury or lower respiratory tract infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or exemplary technical descriptions. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0060] FIG1 is a schematic structural diagram of celastrol I.

[0061] FIG2 is a schematic diagram of the structure of celastrol II.

[0062] FIG3 is a schematic diagram of the structure of celastrol III.

[0063] FIG4 is a schematic diagram of the structure of celastrol A.

[0064] FIG5 is a schematic diagram of the structure of celastrol B.

[0065] FIG6 is a schematic structural diagram of matrine.

[0066] FIG7 is a schematic structural diagram of sophocarpine.

[0067] FIG8 is a schematic structural diagram of cyperine A.

[0068] FIG9 is a schematic structural diagram of cyperine B.

[0069] Figure 10 is a graph showing the anti-inflammatory effects of different pharmaceutical compositions, indicating that each composition can dose-dependently inhibit the excessive production of NO in RAW264.7 mouse macrophages induced by LPS and exert an anti-inflammatory effect, wherein Figure A is a graph showing the anti-inflammatory effect of the composition of Example 3, Figure B is a graph showing the anti-inflammatory effect of the composition of Example 4, Figure C is a graph showing the anti-inflammatory effect of the composition of Example 5, Figure D is a graph showing the anti-inflammatory effect of the composition of Example 6, Figure E is a graph showing the anti-inflammatory effect of the composition of Example 7, and Figure F is a graph showing the anti-inflammatory effect of the composition of Example 8.

[0070] Figure 11 is a bar graph of the results of the NO production inhibition experiment, indicating that the chelidonin components can dose-dependently inhibit the excessive production of NO in LPS-induced RAW264.7 mouse macrophages, wherein Figure A is a bar graph of the NO production inhibition experiment results of chelidonin III, Figure B is a bar graph of the NO production inhibition experiment results of chelidonin A, and Figure C is a bar graph of the NO production inhibition experiment results of chelidonin B.

[0071] Figure 12 is a bar graph of the QR induction activity experimental results, indicating that the chelidonin components have an inhibitory effect on oxidative stress, wherein Figure A is a bar graph of the QR induction activity experimental results of chelidonin III, Figure B is a bar graph of the QR induction activity experimental results of chelidonin A, and Figure C is a bar graph of the QR induction activity experimental results of chelidonin B.

[0072] FIG13 shows the effect of celastrol A, a component of celastrol, on the transcriptome of human bronchial epithelial Beas-2B cells.

[0073] FIG14 shows the effect of scutellarine A, a component of the matrine class, on the transcriptome of human bronchial epithelial Beas-2B cells.

[0074] Figure 15 shows the potential anti-inflammatory functional genes (targets) of clematisin A, a component of the clematisin class, and clematine A, a component of the matrine class.

[0075] Figure 16 is a H&E staining photograph of mouse lung tissue, indicating that the basidioscin components in the pharmaceutical composition, namely basidioscin I (BS1), basidioscin II (BS2) and basidioscin III (BS3), and the matrine component matrine (MR) and the combination of the two components (BS3+MR) can all reduce LPS-induced pathological changes in mouse lung tissue, and the effect of the combination group is more obvious. The concentration of the positive control drug DEX is 1 mg / kg, the concentration of LPS is 10 mg / kg, the concentrations of BS1, BS2, BS3 and MR are all 20 mg / kg, and the concentration of the pharmaceutical composition (BS3+MR) is 20 mg / kg of BS3 and 20 mg / kg of MR.

[0076] Figure 17 is a bar graph of the results of enzyme-linked immunosorbent assay, indicating that the basidiomycin components in the pharmaceutical composition, namely basidiomycin I (BS1), basidiomycin II (BS2) and basidiomycin III (BS3), and the matrine component matrine (MR) and the combination of the two components (BS3+MR) can inhibit the increase of the inflammatory factor IL-1β in the alveolar lavage fluid of mice induced by LPS, among which the activity of the combination is significantly enhanced. The concentration of the positive control drug DEX is 1 mg / kg, the concentration of LPS is 10 mg / kg, the concentrations of BS1, BS2, BS3 and MR are all 20 mg / kg, and the concentration of the pharmaceutical composition (BS3+MR) is 20 mg / kg of BS3 and 20 mg / kg of MR.

[0077] Figure 18 is a scatter plot of the routine blood test results in mice, indicating that the basidiomycin components in the pharmaceutical composition, namely basidiomycin I (BS1), basidiomycin II (BS2) and basidiomycin III (BS3), and the matrine component matrine (MR) and the combination of the two components (KF+MR) can all reduce the number of inflammatory cells in the blood of mice, among which the activity of the composition is significantly enhanced. The concentration of the positive control drug DEX is 1 mg / kg, the concentration of LPS is 10 mg / kg, the concentrations of BS1, AG, BS3 and MR are all 20 mg / kg, and the concentration of the pharmaceutical composition (BS3+MR) is 20 mg / kg of BS3 and 20 mg / kg of MR.

[0078] Figure 19 is a bar graph of the qRT-PCR results, indicating that the basidiomycin components in the pharmaceutical composition, namely basidiomycin I (BS1), basidiomycin II (BS2) and basidiomycin III (BS3), and the matrine component matrine (MR) and the combination of the two components (BS3+MR) can all reduce the increase in the mRNA level of inflammatory factors in the lung tissue of mice, among which the activity of the composition is significantly enhanced. The concentration of the positive control drug DEX is 1 mg / kg, the concentration of LPS is 10 mg / kg, the concentrations of BS1, BS2, BS3 and MR are all 20 mg / kg, and the concentration of the pharmaceutical composition (BS3+MR) is 20 mg / kg of BS3 and 20 mg / kg of MR.

[0079] Figure 20 is a bar graph of Western Blot and qRT-PCR results, indicating that the celery scutellariae components in the pharmaceutical composition can regulate the ferroptosis-related pathway proteins SLC7A11 and GPX4 and their mRNA levels. The concentration of the positive control drug DEX is 1 mg / kg, the concentration of LPS is 10 mg / kg, and the concentrations of BS1, BS2, BS3 and MR are all 20 mg / kg.

[0080] FIG21 is a photograph of H&E staining of mouse lung tissue, indicating that both celastrol components and matrine components can attenuate CS-induced lung pathological damage in mice.

[0081] FIG22 is a bar graph of the results of enzyme-linked immunosorbent assay, which shows that both celery alkaloid components and matrine components can inhibit the production of CS-induced inflammatory factors IL-1β and TNF-α in the lungs of mice.

[0082] FIG23 is a bar graph showing the results of routine blood tests in mice, indicating that both the celery extract components and the matrine components reduced the number of inflammatory cells in the peripheral blood of mice induced by CS.

[0083] FIG24 is a bar graph of flow cytometry results, indicating that celastrol fractions and matrine fractions inhibit the proportion of Th1 cells in CS-induced mouse lung tissue and improve the balance between Th1 and Th2.

[0084] FIG25 is a bar graph of flow cytometry results, indicating that celastrol fractions and matrine fractions inhibit the proportion of Th17 cells in CS-induced mouse lung tissue and improve the balance between Th17 and Treg.

[0085] Figure 26 is a bar graph of the results of improving intestinal flora, indicating that the chelidonin components can improve the composition of intestinal flora, among which the chelidonin components mainly improve the murinus species in the Lactobacillus genus, while the matrine components mainly play a role in improving COPD through the caecimuris and vulgatus species in the Bacteroides genus.

[0086] Figure 27 is a line graph of cell viability, demonstrating that the combination of basidioscin III (BS3) and basidioscin A (BSA) with matrine (MR), a matrine-like component, can reduce the toxicity of MR to liver cells. The ratio of BS3 to basidioscin and MR is 1:1.

[0087] Figure 28 is a line graph of cellular ROS levels, demonstrating that the combination of basidioscin III (BS3) and basidioscin A (BSA) with matrine (MR), a matrine-like component, can reduce MR's ability to induce ROS in liver cells. The ratio of BS3 and BSA to MR is 1:1.

[0088] Figure 29 shows the 1 H NMR spectrum.

[0089] Figure 30 is a diagram of celastrol A 13 C NMR spectrum.

[0090] Figure 31 is the 1 H NMR spectrum.

[0091] Figure 32 shows the 13 C NMR spectrum.

[0092] Figure 33 is a diagram of cyperine A 1 H NMR spectrum.

[0093] Figure 34 is a diagram of cyperine A 13 C NMR spectrum.

[0094] Figure 35 is a diagram of cyperine B 1 H NMR spectrum.

[0095] Figure 36 is a diagram of cyperine B 13 C NMR spectrum. DETAILED DESCRIPTION

[0096] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0097] It should be noted that when a component is referred to as being "fixed on" or "disposed on" another component, it may be directly on the other component or indirectly on the other component. When a component is referred to as being "connected to" another component, it may be directly or indirectly connected to the other component. The terms "upper", "lower", "left", "right", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of description and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on this application. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to the specific circumstances. The terms "first" and "second" are only used for the purpose of convenience of description and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features. "Multiple" means two or more, unless otherwise clearly and specifically defined.

[0098] In order to illustrate the technical solution provided by this application, a detailed description is given below with reference to specific drawings and embodiments.

[0099] In a first aspect, the present invention provides a pharmaceutical composition for treating pulmonary inflammatory diseases, comprising the following components:

[0100] 1-100 parts of celery scutellariae components,

[0101] 1-20 parts of matrine components;

[0102] The general structural formula of the celeriac component is shown in Formula A, which is:

[0103] wherein R4 is H or a group; R1, R2, R3, and R5 are independently selected from one of the following groups: H, CH3, CH3CO, glucosyl, rhamnosyl, glucose rhamnosyl, glucose glucosyl, xylosyl, and sucrose;

[0104] The general structural formula of the matrine component is shown in Formula B:

[0105] Wherein, R6, R7, and R8 are independently selected from one of the following groups: H, OH, OCH3, and CH3COO.

[0106] The first beneficial effect provided by the embodiments of the present application is that: the pharmaceutical composition provided by the present application mainly includes celastrol components and matrine components. The celastrol components have QR inducing activity, can inhibit LPS-induced excessive production of NO in RAW264.7 mouse macrophages, reduce LPS-induced pathological changes in mouse lung tissue, inhibit the increase of inflammatory factor IL-1β in the alveolar lavage fluid of mice induced by LPS, and can reduce the number of inflammatory cells in the blood of mice, thereby exerting a lung protective effect and alleviating lung inflammation.

[0107] Among them, the effects of chelidonin and matrine components on the transcriptome of human bronchial epithelial Beas-2B cells showed that there were significant differences in the inflammatory regulatory genes regulated by chelidonin and matrine, and the anti-inflammatory mechanisms of the two components were different.

[0108] The synergistic effect of the celeriac components and the matrine components in the pharmaceutical composition, the two of which play a role in treating lung diseases through different mechanisms of action. The present application discovered a new mechanism by which celeriac components treat acute lung injury by regulating ferroptosis-related pathway proteins SLC7A11 and GPX4. At the same time, matrine components can inhibit the excessive activation of NLRP3 inflammasomes and inhibit NF-kB activation. In addition, celeriac components and matrine components have a regulatory effect on different intestinal flora, among which celeriac components mainly improve the abundance of murinus in Lactobacillus, while matrine components mainly improve the abundance of caecimuris and vulgatus in Bacteroides, and it is found that the changes in strains are correlated with the expression of immune factors. celeriac components and matrine components synergistically exert lung protective effects through different mechanisms of action.

[0109] The binding of scutellarin and matrine components to key inflammatory regulatory proteins is studied. The newly discovered scutellarin components, scutellarin A and B, have far superior binding abilities to iNOS and COX-2 than the known scutellarin component, scutellarin III. The new matrine component, scutellarin B, also has superior binding abilities to iNOS than the known matrine components, matrine and sophocarpine. The newly discovered scutellarin and matrine components exhibit enhanced anti-inflammatory effects, and these newly discovered compounds are also highly promising for development.

[0110] The chelidonin components have a reducing effect on high-concentration matrine components. The chelidonin components can inhibit the hepatotoxicity and renal toxicity of matrine components and inhibit the ROS generation caused by matrine components.

[0111] Furthermore, it is described that the matrine components can increase the solubility of the scutellariae components in water, thereby increasing the water solubility and thus improving the bioavailability.

[0112] In addition, the interaction between the scutellariae and matrine components of "enhancing efficacy and reducing toxicity, complementary mechanisms, and mutual solubility" is described, as well as the necessity and value of the two types of components forming a pharmaceutical composition.

[0113] In some embodiments, the pharmaceutical composition comprises the following components:

[0114] 60-66 parts of the celeriac component,

[0115] The matrine component is 2-5 parts.

[0116] In some embodiments, the lycoside component is selected from at least one of lycoside I, lycoside II, lycoside III, lycoside A, and lycoside B;

[0117] In some embodiments, the matrine component is selected from at least one of matrine, sophoracarpine, lycopodipine A, and lycopodipine B.

[0118] In some embodiments, the pharmaceutical composition comprises the following components:

[0119] In some embodiments, the pharmaceutical composition comprises the following components:

[0120] 10-15 parts of the described celery scutellarin III,

[0121] 1 part of described matrine.

[0122] In some specific embodiments, the pharmaceutical composition comprises the following components:

[0123] 12 parts of the described celery scutellarin III,

[0124] 1 part of described matrine.

[0125] In some embodiments, the method for extracting celastrol A and celastrol B comprises the following steps:

[0126] The dried whole herb of Herba Lycoris Radiatae is crushed, soaked at 20-40°C for 1-10 hours, and then heated and refluxed with 60-80% ethanol to obtain an extract; the extract is filtered and concentrated in sequence to obtain a total extract;

[0127] After the total extract was suspended in water, it was extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain various organic extracts;

[0128] The ethyl acetate extract is separated by normal phase silica gel column chromatography, wherein first, gradient elution is performed using an eluent of petroleum ether and ethyl acetate in a volume ratio of 0.1-1:0.1-1, and then gradient elution is performed using an eluent of ethyl acetate / methanol in a volume ratio of 0.1-1:0.1-1; 8 fractions are collected in chronological order to obtain first elution products E1-E8;

[0129] After the first elution product E7 fraction was passed through an MCI column to remove the pigment, gradient elution was performed using a methanol / water eluent with a volume ratio of 1-3:0.1-7, and 8 fractions were collected in chronological order to obtain the second elution products E7a-h;

[0130] The second eluted product E7d was separated by a normal silica gel column using a gradient elution of dichloromethane / methanol in a volume ratio of 0.1 to 30:1. Eight fractions were collected in chronological order to obtain the third eluted products E7a-h.

[0131] The third elution product E7d5 was separated and purified by Sephadex LH-20 and C18 reverse phase silica gel to obtain compounds lycoside A (purity>98%) and lycoside B (purity>98%).

[0132] Wherein, the structural formula of the celastrol A is as follows:

[0133] The structural formula of the celery scutellarin B is as follows:

[0134] Among them, the total extract is obtained by concentrating the alcohol extract. In order to enable the subsequent extraction process to better extract the extract into several parts with different polarities, the ethanol is required to be basically recovered during concentration. At this time, the total extract is very thick and cannot be extracted. Therefore, it is necessary to use water with high polarity to disperse it, and then use solvents with increasing polarity to extract it in turn.

[0135] In some specific embodiments, when Sephadex LH-20 is used for separation, the elution program of methanol / water gradient elution is: 3:7→4:6.

[0136] In some embodiments, the extraction method of the lycopodium alpinum A and the lycopodium alpinum B is:

[0137] The whole herb of Herba Lycopodii is ground and extracted with a 0.1-0.5 mol / L hydrochloric acid aqueous solution (after acid-water extraction, alkaloids are dissolved in the acid water in the form of salts, and adding alkali precipitates the alkaloids, which facilitates sufficient extraction with dichloromethane). The extract is adjusted to alkaline, and then repeatedly extracted with dichloromethane. The total alkaloid extract is obtained after concentration under reduced pressure.

[0138] The total alkaloid extract was separated by a normal phase silica gel column and eluted with a petroleum ether / dichloromethane / ammonia water eluent in a volume ratio of 1:1:0.02-0.03; then gradient elution was performed with a dichloromethane / methanol eluent in a volume ratio of 6-97:3-4, and 10 fractions were collected in chronological order to obtain the fourth elution products S1-S10;

[0139] The fourth eluted product S5 was purified by Sephadex LH-20 column chromatography using methanol as eluent, and the purified S5 was separated by HPLC using a mobile phase having a pH of 3 to 4 and a methanol content of 5% to 10% by volume;

[0140] After impurities were removed by macroporous adsorption resin column, lycopodium B (purity>98%) and lycopodium A (purity>98%) were obtained;

[0141] Wherein, the structural formula of the cyperine A is as follows:

[0142] The structural formula of the lycopodipine B is as follows:

[0143] In some embodiments, the extraction process of the lycopodiola rosea I, the lycopodiola rosea II, and the lycopodiola rosea III comprises the following steps:

[0144] The dried whole herb of Herba Lycopodii is crushed and soaked at 20-40°C for 1-10 hours, and then subjected to heating and reflux extraction with a 60-80% by weight ethanol aqueous solution; the extract is filtered and concentrated to obtain a total extract;

[0145] After the total extract was suspended in water, it was extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain various organic extracts;

[0146] The ethyl acetate extract was added with water and passed through an MCI column, and then eluted with methanol-water solutions with a mass percentage of 20%-30%, 40%-50%, and 60%-70% in sequence to obtain the first fraction, the second fraction, and the third fraction in sequence;

[0147] The first fraction is then eluted with pure water, ethanol-water solutions with a mass percentage of 20%-30%, 40%-50%, 60%-70%, and pure ethanol, to obtain component a, component b, component c, component d, and component e, respectively;

[0148] wherein the component b is purified by gel chromatography using methanol as the eluent and recrystallized to obtain celastrol I (purity>98%);

[0149] The component C was purified by gel chromatography using methanol as the eluent, and recrystallized to obtain celastrol II (purity>98%);

[0150] The third fraction was purified by gel chromatography using methanol as eluent and recrystallized to obtain celastrol III (purity>98%).

[0151] The MCI column is an MCI gel column, which is a small-pore resin gel column (polystyrene-based reverse-phase resin filler), the macroporous resin column is an AB-8 macroporous resin column, and the gel chromatography column is a Sephadex LH-20 gel chromatography column.

[0152] A second aspect of an embodiment of the present application provides a preparation for inflammatory lung diseases, wherein the mass percentage of the above-mentioned pharmaceutical composition as an active ingredient in the preparation is 0.1%-99.9%, and the rest is a pharmaceutically acceptable excipient; wherein the dosage form of the preparation is tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, lozenges, granules, electuary, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, drops or patches.

[0153] The second beneficial effect provided by the embodiments of the present application is that: a preparation for inflammatory lung diseases is provided, wherein the mass percentage of the above-mentioned pharmaceutical composition as an effective component in the preparation is 0.1%-99.9%; therefore, the provided preparation has the effect of alleviating lung inflammation, protecting the lungs, has excellent anti-inflammatory effects, and is conducive to wide application.

[0154] The composition of the present application is prepared into a unit dose pharmaceutical preparation form, where the unit dose form refers to the unit of preparation, such as each tablet, each capsule, each bottle of oral solution, each bag of granules, etc.

[0155] Preferably, the preparation is in the form of capsules, tablets, oral liquids, granules, pills, powders, pills or pastes.

[0156] The preparation for oral administration may contain common excipients, binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavorings and wetting agents, and the tablets may be coated if necessary.

[0157] Suitable excipients include lactose, D-mannitol, D-sorbitol, starches such as α-starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminum silicate, etc. Suitable binders include α-starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, sugar, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, pyrrolidone, etc. Suitable fillers include cellulose, mannitol, lactose, and other similar fillers. Suitable disintegrants include lactose, sugar, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, sodium aminoalkyl, sodium carboxymethyl starch, light anhydrous silicic acid, polyvinyl pyrrolidone, and sodium starch glycolate. Suitable lubricants include magnesium stearate, calcium stearate, talc, silica gel, sodium lauryl sulfate, etc. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate. Suitable coating agents include hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, and the like. Suitable colorants include water-soluble edible citron yellow dyes (e.g., edible red No. 2 and No. 3, edible yellow No. 4 and No. 5, edible blue No. 1 and No. 2); water-insoluble precipitating dyes (e.g., aluminum salts of the aforementioned water-soluble edible citron yellow dyes); and natural dyes (e.g., β-carotene, chlorophyll, and ferric red). Suitable sweeteners include sodium saccharin, glycyrrhetinic acid, aspartame, and stevia.

[0158] For injections, a liquid unit dosage form is prepared containing the active substance of the present invention and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. Solutions are typically prepared by dissolving the active substance in a carrier, sterilizing by filtration before filling into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservatives, and buffers may also be dissolved in the carrier. To improve stability, the composition can be frozen after filling into the vial, and the water removed under vacuum.

[0159] The composition of the present application can be optionally added with a suitable pharmaceutically acceptable carrier when it is prepared into a medicament. The pharmaceutically acceptable carrier is selected from the group consisting of mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, mercaptoacetic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose glucose, fructose, dextrose, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, monocyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc.

[0160] The third aspect of the embodiments of the present application provides the use of the above-mentioned pharmaceutical composition for treating inflammatory lung diseases in the preparation of drugs for chronic obstructive pulmonary disease, acute lung injury or lower respiratory tract infection.

[0161] The third beneficial effect provided by the embodiments of the present application is that the pharmaceutical composition for treating inflammatory lung diseases has excellent anti-inflammatory effects, can effectively reduce lung inflammation, and can be widely used in the preparation of drugs for chronic obstructive pulmonary disease, acute lung injury or lower respiratory tract infection.

[0162] The following provides specific examples for illustration.

[0163] The compounds of lycopodiola I, lycopodiola II and lycopodiola III described in this application are known lycopodiola compounds. 1 H NMR and 13 The structure was determined by C NMR spectrum and literature comparison.

[0164] The present application will be further described below with reference to the embodiments.

[0165] Example 1:

[0166] This example lists the preparation methods of 5 representative scutellarin components and 2 representative matrine components. The details are as follows:

[0167] The celastrol I, celastrol II, and celastrol III provided in this application can be prepared by the following method:

[0168] (1) Preparation of Herba Lycopodii Extract

[0169] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with 75% ethanol for 3 times, each time for 3 hours. After filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0170] (2) Extraction

[0171] The total extract was suspended in water and extracted four times with equal volumes of petroleum ether, dichloromethane and ethyl acetate to obtain organic layers.

[0172] (3) Impurity removal

[0173] The ethyl acetate layer was dissolved in water and passed through an MCI column. Elution with 30%, 50%, and 70% methanol-water removed the pigment and some impurities. The 30% methanol-water fraction (A) and the 70% methanol-water fraction (B) were used for further separation.

[0174] (4) Refining

[0175] Fraction A was separated by AB-8 macroporous resin column chromatography and gradient eluted with pure water, 30% ethanol-water, 50% ethanol-water, 70% ethanol-water, and pure ethanol for 3 column volumes each. The 30% ethanol-water fraction (C) and the 50% ethanol-water fraction (D) were used for further purification. Fraction C was purified by Sephadex LH-20 gel chromatography using methanol as the eluent and finally recrystallized at 4°C using methanol as the solvent to obtain high-purity cyperus glomerulone I with a purity of 99% and a yield of 82%.

[0176] The D fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent, and finally recrystallized to obtain high-purity cyperus scutellarin II with a purity of 99% and a yield of 78%.

[0177] The B fraction was purified by Sephadex LH-20 gel chromatography column with methanol as eluent, and finally recrystallized to obtain high-purity cyperus scutellarin III with a purity of 99% and a yield of 85%.

[0178] The scutellarin A and scutellarin B in the scutellaria baicalensis provided in this application can be prepared by the following method:

[0179] (1) Preparation of Herba Lycopodii Extract

[0180] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with 75% ethanol under heating and reflux for 3 times, each time for 3 hours; after filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0181] (2) Extraction

[0182] The total extract was suspended with water at a volume ratio of 1:5, and extracted four times with petroleum ether, dichloromethane and ethyl acetate in a volume 6 times that of the total extract to obtain organic layers.

[0183] (3) Refining

[0184] The ethyl acetate fraction was first separated by normal phase silica gel column chromatography using petroleum ether / ethyl acetate (1:0→0:1) and ethyl acetate / methanol (1:0→0:1) as eluents to obtain 8 fractions (E1-E8); the E7 fraction was first subjected to MCI column chromatography to remove some of the pigment using methanol / water (3:7→1:0) as eluent to obtain 8 fractions (E7a-h); the E7d fraction was separated by normal phase silica gel column chromatography (dichloromethane / methanol, 30:1→0:1) to obtain 6 fractions (E7d1-6); the E7d5 fraction was separated and purified using Sephadex LH-20 gel and C18 reverse phase silica gel to obtain compounds celastrol A (purity 99%, yield 68%) and celastrol B (purity 99%, yield 71%).

[0185] The preparation method of lycopodipine A and lycopodipine B in lycopodipine provided in the present application comprises the following specific steps:

[0186] (1) Preparation of Herba Lycopodii Total Alkaloids

[0187] A batch of dried and crushed whole herbal medicine of Herba Lycopodii was extracted with a 0.3 mol / L aqueous hydrochloric acid solution; the extract was adjusted to pH 10 with a 1.32 mol / L aqueous sodium hydroxide solution, then extracted four times with dichloromethane, and concentrated under reduced pressure to obtain a total alkaloid extract.

[0188] (2) Refining

[0189] The total alkaloid extract was separated by normal phase silica gel column, and petroleum ether / dichloromethane / ammonia (1:1:0.03) and dichloromethane / methanol (97:3→6:4) were used as eluents to obtain 10 fractions (S1-S10). Fractions S5-S7 were selected for further system separation; fraction S5 was purified by C 18 Reverse-phase silica gel column chromatography (methanol / water, 0:1→1:0) yielded six fractions (S5a-f). Fraction S5f was first purified by Sephadex LH-20 column chromatography using pure methanol as eluent. The purified sample was further separated on C18 reverse-phase silica gel to obtain compounds scutellarine A (99% purity, 65% yield) and scutellarine B (99% purity, 70% yield).

[0190] Comparative Example 1

[0191] Cleomedin I, Cleomedin II, and Cleomedin III can be prepared by the following method:

[0192] (1) Preparation of Herba Lycopodii Extract

[0193] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with 75% ethanol for 3 times, each time for 3 hours. After filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0194] (2) Extraction

[0195] The total extract was suspended in water and extracted four times with equal volumes of petroleum ether, dichloromethane and ethyl acetate to obtain organic layers.

[0196] (3) Impurity removal

[0197] The ethyl acetate layer was dissolved in water and passed through an MCI column. Elution with 30%, 50%, and 70% methanol-water removed the pigment and some impurities. The 30% methanol-water fraction (A) and the 70% methanol-water fraction (B) were used for further separation.

[0198] (4) Refining

[0199] Fraction A was separated by D101 macroporous resin column chromatography and gradient eluted with pure water, 30% ethanol-water, 50% ethanol-water, 70% ethanol-water, and pure ethanol for 3 column volumes each. The 30% ethanol-water fraction (C) and the 50% ethanol-water fraction (D) were used for further purification. Fraction C was purified by Sephadex LH-20 gel chromatography using methanol as the eluent and finally recrystallized at 4°C using methanol as the solvent to obtain high-purity cyperus glomerulone I with a purity of 99% and a yield of 72%.

[0200] The D fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent, and finally recrystallized to obtain high-purity cyperus scutellarin II with a purity of 99% and a yield of 76%.

[0201] The B fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent, and finally recrystallized to obtain high-purity cyperus scutellarin III with a purity of 99% and a yield of 83%.

[0202] Comparative Example 2

[0203] Cleomedin I, Cleomedin II, and Cleomedin III can be prepared by the following method:

[0204] (1) Preparation of Herba Lycopodii Extract

[0205] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with water 3 times, each time for 3 hours; after filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0206] (2) Extraction

[0207] The total extract was suspended in water and extracted four times with equal volumes of petroleum ether, dichloromethane and ethyl acetate to obtain organic layers.

[0208] (3) Impurity removal

[0209] The ethyl acetate layer was dissolved in water and passed through an MCI column. Elution with 30%, 50%, and 70% methanol-water removed the pigment and some impurities. The 30% methanol-water fraction (A) and the 70% methanol-water fraction (B) were used for further separation.

[0210] (4) Refining

[0211] Fraction A was separated by AB-8 macroporous resin column chromatography and gradient eluted with pure water, 30% ethanol-water, 50% ethanol-water, 70% ethanol-water, and pure ethanol for 3 column volumes each. The 30% ethanol-water fraction (C) and the 50% ethanol-water fraction (D) were used for further purification. Fraction C was purified by Sephadex LH-20 gel chromatography using methanol as the eluent and finally recrystallized at 4°C using methanol as the solvent to obtain high-purity cyperus scutellarin I with a purity of 99% and a yield of 64%.

[0212] The D fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent, and finally recrystallized to obtain high-purity cyperus scutellarin II with a purity of 99% and a yield of 62%.

[0213] The B fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent, and finally recrystallized to obtain high-purity cyperus scutellarin III with a purity of 98% and a yield of 51%.

[0214] Comparative Example 3

[0215] Cleomedin I, Cleomedin II, and Cleomedin III can be prepared by the following method:

[0216] (1) Preparation of Herba Lycopodii Extract

[0217] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with 75% ethanol for 3 times, each time for 3 hours. After filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0218] (2) Extraction

[0219] The total extract was suspended in water and extracted four times with equal volumes of petroleum ether, dichloromethane and ethyl acetate to obtain organic layers.

[0220] (3) Impurity removal

[0221] The ethyl acetate layer was dissolved in water and passed through an MCI column. Elution with 30%, 50%, and 70% methanol-water removed the pigment and some impurities. The 30% methanol-water fraction (A) and the 70% methanol-water fraction (B) were used for further separation.

[0222] (4) Refining

[0223] Fraction A was separated by AB-8 macroporous resin column chromatography and gradient eluted with pure water, 30% ethanol-water, 50% ethanol-water, 70% ethanol-water, and pure ethanol for 3 column volumes each. The 30% ethanol-water fraction (C) and the 50% ethanol-water fraction (D) were used for further purification. Fraction C was purified by Sephadex LH-20 gel chromatography using methanol as the eluent to obtain celastrol I with a purity of 83% and a yield of 89%.

[0224] The D fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent to obtain cyperus glomerulone II with a purity of 76% and a yield of 85%.

[0225] The B fraction was purified by Sephadex LH-20 gel chromatography using methanol as the eluent to obtain celastrol III with a purity of 68% and a yield of 90%.

[0226] Comparative Example 4

[0227] Cleomedin A and Cleomedin B can be prepared by the following method:

[0228] (1) Preparation of Herba Lycopodii Extract

[0229] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with 75% ethanol under heating and reflux for 3 times, each time for 3 hours; after filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0230] (2) Extraction

[0231] The total extract was suspended with water at a volume ratio of 1:5, and extracted four times with petroleum ether, dichloromethane and ethyl acetate in a volume 6 times that of the total extract to obtain organic layers.

[0232] (3) Refining

[0233] The ethyl acetate fraction was first separated by normal phase silica gel column chromatography using petroleum ether / ethyl acetate (1:0→0:1) and ethyl acetate / methanol (1:0→0:1) as eluents to obtain 8 fractions (E1-E8); the E7 fraction was first subjected to MCI column chromatography to remove some of the pigment using methanol / water (3:7→1:0) as eluent to obtain 8 fractions (E7a-h); the E7d fraction was separated by normal phase silica gel column chromatography (dichloromethane / methanol, 30:1→0:1) to obtain 6 fractions (E7d1-6); the E7d5 fraction was separated and purified by Sephadex LH-20 gel to obtain compounds celastrol A (purity 67%, yield 88%) and celastrol B (purity 71%, yield 83%).

[0234] Comparative Example 5

[0235] Cleomedin A and Cleomedin B can be prepared by the following method:

[0236] (1) Preparation of Herba Lycopodii Extract

[0237] The dried whole herb of Herba Lycopodii was crushed and soaked at room temperature for 3 hours, and then extracted with water under reflux for 3 times, each time for 3 hours. After filtering the extract, the extract was vacuum concentrated on a rotary evaporator to obtain a total extract.

[0238] (2) Extraction

[0239] The total extract was suspended with water at a volume ratio of 1:5, and extracted four times with petroleum ether, dichloromethane and ethyl acetate in a volume 6 times that of the total extract to obtain organic layers.

[0240] (3) Refining

[0241] The ethyl acetate fraction was first separated by normal phase silica gel column chromatography using petroleum ether / ethyl acetate (1:0→0:1) and ethyl acetate / methanol (1:0→0:1) as eluents to obtain 8 fractions (E1-E8); the E7 fraction was first subjected to MCI column chromatography to remove some of the pigment using methanol / water (3:7→1:0) as eluent to obtain 8 fractions (E7a-h); the E7d fraction was separated by normal phase silica gel column chromatography (dichloromethane / methanol, 30:1→0:1) to obtain 6 fractions (E7d1-6); the E7d5 fraction was separated and purified by Sephadex LH-20 gel to obtain compounds celastrol A (purity 65%, yield 46%) and celastrol B (purity 58%, yield 41%).

[0242] Comparative Example 6

[0243] Lycopodipine A and Lycopodipine B can be prepared by the following method:

[0244] (1) Preparation of Herba Lycopodii Total Alkaloids

[0245] A batch of dried and crushed whole herbal medicine of Herba Lycopodii was extracted with a 0.3 mol / L aqueous hydrochloric acid solution; the extract was adjusted to pH 10 with a 1.32 mol / L aqueous sodium hydroxide solution, then extracted four times with dichloromethane, and concentrated under reduced pressure to obtain a total alkaloid extract.

[0246] (2) Refining

[0247] The total alkaloid extract was separated by normal phase silica gel column, and petroleum ether / dichloromethane / ammonia (1:1:0.03) and dichloromethane / methanol (97:3→6:4) were used as eluents to obtain 10 fractions (S1-S10). Fractions S5-S7 were selected for further system separation; fraction S5 was purified by C 18 Reverse-phase silica gel column chromatography (methanol / water, 0:1→1:0) yielded six fractions (S5a-f). Fraction S5f was purified by Sephadex LH-20 column chromatography using pure methanol as eluent to obtain scutellarine A (purity 67%, yield 79%) and scutellarine B (purity 72%, yield 76%).

[0248] Example 2:

[0249] Structural determination data of the compound prepared in Example 1:

[0250] This example lists the structural determination data of 5 representative scutellarin components and 2 representative matrine components, including the structural identification of 4 new compounds, as follows:

[0251] (1) Structural identification of celastrol A

[0252] The compound is a yellow oil that is soluble in methanol. Using dichloromethane / methanol as the developing solvent at a ratio of 4:1 and adding a drop of glacial acetic acid, thin-layer chromatography was performed. Dark spots appeared under a 254nm UV lamp. Yellow spots were observed by using a p-anisaldehyde-concentrated sulfuric acid solution and heating for color development, indicating that it is a flavonoid. f The value is 0.38. HRESIMS shows that its quasi-molecular ion [M+H] + Peak m / z 879.1991 (calculated value 879.1978), the molecular formula is deduced to be C 42 H 38 O 21 .

[0253] 1 The H NMR (600 MHz, CD3OD) spectrum showed two groups of AA′BB′ spin coupling systems, namely B ring [δ H7.35 (2H, d, J = 8.9 Hz) and 6.78 (2H, d, J = 8.9 Hz)] and E ring [δ H 8.12 (2H, d, J = 9.0 Hz) and 6.90 (2H, d, J = 9.0 Hz)]. In addition, there is a trisubstituted aromatic ring A ring [δ H 5.96 (1H, d, J = 2.1 Hz) and 5.90 (1H, d, J = 2.1 Hz)] and an aromatic proton signal on the D ring δ H 6.57 (1H, s). There are two sugar anomeric proton signals δ H The presence of two sugars was suggested by the presence of 5.74 (1H, d, J = 7.7 Hz) and 5.23 (1H, d, J = 1.9 Hz). Furthermore, the presence of multiple overlapping peaks between chemical shift values ​​of 3 and 4 further confirmed the presence of two sugars. 13 The C NMR (150 MHz, CD3OD) spectrum showed 38 carbon signals, and two keto carbonyl signals (δ C 192.5,179.6), two double bond carbon signals (δ C 158.8,134.7) and the presence of twenty-four aromatic carbon signals. In addition, the carbon signal δ C 119.5 and δ C 81.7 is the signal of two oxygen-linked carbons, and based on the number of oxygen atoms in the molecular formula, it is believed to be an alkylene oxide structure formed by two carbon atoms at the C-2 and C-3 positions.

[0254] Further, through the correlation between H-2′ and C-2 and H-2″′ and C-2″ in the HMBC spectrum (Figure 2-1), it was determined that ring B is connected to ring C, and ring D is connected to ring E. From this, it was concluded that the compound is a biflavonoid glycoside formed by a kaempferol-type structure (ring A, ring B and ring C), an epoxyflavanone-type structure and two sugars. C-3 and C-8″ are the only possible connection modes of the two flavonoids. Further, based on HSQC, 1 H- 1 H COSY and HMBC assigned two glycosyl signals, namely β-glucose [δ H 5.74 (1H, d, J = 7.7 Hz), 3.73 (1H, d, J = 12.0, 2.1 Hz), 3.63 (1H, dd, J = 9.2, 7.7 Hz), 3.55 (1H, dd, J = 9.2, 8.7 Hz), 3.49 (1H, dd, J = 12.0, 5.9 Hz), 3.27 (1H, dd, J = 9.8, 8.7 Hz) and 3.23 (1H, ddd, J = 9.8, 5.9, 2.1 Hz), as well as δ C100.1, 79.7, 79.0, 71.9, 78.5 and 62.9] and α-rhamnose [δ H 5.23 (1H, d, J = 1.9 Hz), 3.98 (1H, dd, J = 3.4, 1.9 Hz), 3.98 (1H, overlap), 3.73 (1H, dd, J = 7.8, 1.7 Hz), 3.32 (1H, overlap) and 0.91 (3H, d, J = 6.2 Hz) and δ C The HMBC spectrum showed that the H-1″″ / C-3″ and H-1″″′ / C-2″″ correlations confirmed the connection mode of the two sugars. The relative configuration of the compound was thus inferred. The structure of this compound is similar to that of balsamiside A reported in the literature, with the main difference being the different connection positions of the two sugars.

[0255] C-2 and C-3 are two chiral carbon atoms, and their absolute configuration was determined by comparing the ECD spectrum of the compound with that of the literature. The ECD spectrum showed a positive cotton effect at 320 nm and a negative cotton effect at 288 nm. The biflavonoid structure of this compound is identical to that of the hydrolysis product of balsamiside A reported in the literature, and its cotton effect is also essentially the same, thus determining its absolute configuration to be 2S, 3S. In summary, the structure of this compound is [(2S,3S)-2,3-epoxy-5,7,4′-trihydroxyflavanone]-(3→8)-kaempferol3″-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside, named kaempferol A.

[0256] (2) Structural identification of celery scutellarin B

[0257] The compound is a yellow oil that is soluble in methanol. Using dichloromethane / methanol as the developing solvent at a ratio of 4:1 and adding a drop of glacial acetic acid, thin-layer chromatography was performed. Dark spots appeared under a 254nm UV lamp. Yellow spots were observed by using a p-anisaldehyde-concentrated sulfuric acid solution and heating for color development, indicating that it is a flavonoid. f The value is 0.38. HRESIMS shows that its quasi-molecular ion [M+H] + Peak m / z 879.1991 (calculated value 879.1978), the molecular formula is deduced to be C 42 H 38 O 21. The 1D NMR and 2D NMR spectra of the compound are basically consistent with those of kaempferol A, confirming that they have the same planar structure and should be a pair of diastereomers. The only difference between the two is the absolute configuration of C-2 and C-3. The ECD spectrum shows that the compound has a negative cotton effect at 320nm and a positive cotton effect at 288nm, which is opposite to that of kaempferol A, indicating that the two compounds have opposite absolute configurations at C-2 and C-3. In summary, the structure of the compound is [(2R,3R)-2,3-epoxy-5,7,4′-trihydroxyflavanone]-(3→8)-kaempferol3″-O-α-L-rhamnopyranosyl-(1→2)-β-D-gluco-pyranoside, named kaempferol B.

[0258] The NMR spectrum data of celastrol A and celastrol B are shown in the following table:

[0259] Table 1. The content of celery scutellarin A and celery scutellarin B 1 H (600MHz) and 13 C(150MHz) data (CD3OD)

[0260] (3) Structural identification of cyperine A

[0261] The compound is a colorless solid soluble in methanol. Using petroleum ether / ethyl acetate as the developing solvent at a ratio of 1:1 and adding a drop of glacial acetic acid, thin layer chromatography was performed. Dark spots appeared under a 254 nm UV lamp. Color development was performed using a modified potassium bismuth iodide reagent, and orange-red spots were observed. f The value is 0.34. HRESIMS shows that its quasi-molecular ion [M+H] + Peak m / z 247.1805 (calculated value is 247.1805), the molecular formula is deduced to be C 15 H 22 N2O, unsaturation degree is 6. 1 The H NMR (600 MHz, CD3OD) spectrum showed sixteen proton signals on eight methylene groups. H3.46 (1H, overlap) and 2.89 (1H, overlap); 1.97 (1H, overlap) and 1.72 (1H, overlap); 1.85 (1H, m) and 1.80 (1H, overlap); 2.01 (1H, overlap) and 1.72 (1H, overlap); 2.07 (1H, overlap) and 1.72 (1H, overlap); 3.46 (1H, overlap) and 2.89 (1H, overlap); 2.79 (1H, m) and 2.34 (1H, m) and 4.25 (1H, dd, J = 14.0, 5.0 Hz) and 3.23 (1H, dd, J = 14.0, 12.9 Hz), four methine proton signals δ H 4.13 (1H, ddd, J = 11.8, 8.4, 7.3 Hz), 3.39 (1H, m), 2.11 (1H, overlap) and 2.07 (1H, overlap), as well as two olefin proton signals δ H 6.64 (1H, ddd, J = 9.8, 4.7, 3.8 Hz) and 5.84 (1H, dt, J = 9.8, 2.0 Hz). 13 The C NMR (150 MHz, CD3OD) spectrum and DEFT spectrum showed 15 carbon signals, including eight methylene carbon signals δ C 57.0, 56.9, 42.0, 28.0, 26.8, 25.2, 20.2 and 19.8, four methine carbon signals δ C 64.6, 52.0, 41.2 and 34.8, two double bond carbon signals δ C 140.7, 124.1 and a carbonyl signal δ C 167.6. 1 H- 1 The position of the α,β-unsaturated ketone was determined by combining the H-14 / H-13 / H2-12 / H-11 related signals in the H COSY spectrum with the H-14 / C-15, H-13 / C-15, and H-11 / C-13 and C-15 related signals in the HMBC spectrum. 1 H- 1The related signals of H-11 / H-7 / H-6 / H-5 / H2-17; H-7 / H2-8 / H2-9 / H2-10 and H-5 / H2-4 / H2-3 / H2-2 in the H COSY spectrum, combined with the related signals of H-11 / C-15; H-17 / C-15; H-17 / C-11; H2-2 / C-6; H2-10 / C-6 and H2-2 / C-10 in the HMBC spectrum, confirmed that the basic skeleton of this compound is a matrine-type alkaloid, and its planar structure is the same as that of sophocarpine. The NOESY spectrum showed signals related to H-17b / H-7 and H-11; H-3b / H-5 and H-6; H-6 / H-8b; and H-11 / H-8a, indicating that H-7 and H-11 are in the β-configuration and H-5 and H-6 are in the α-configuration, thus confirming the relative configuration of the compound. Based on the relative configuration, the compound is identified as a new compound.

[0262] The compound's C-5, C-6, C-7, and C-11 positions are all chiral carbon atoms, and its absolute configurations are possible in two forms: 5R, 6R, 7R, 11S and 5S, 6S, 7S, 11R. Using the ECD calculation method, ECD spectra of the two configurations were obtained. The calculated ECD curve for the 5S, 6S, 7S, 11R configuration was found to be consistent with the experimentally measured ECD curve of the compound. Therefore, the absolute configuration was determined to be 5S, 6S, 7S, 11R, and the compound was named cyperine A.

[0263] (4) Structural identification of cyperine B

[0264] The compound is a colorless solid soluble in methanol. Using petroleum ether / ethyl acetate as the developing solvent at a ratio of 1:1 and adding a drop of glacial acetic acid, thin layer chromatography was performed. No dark spots were observed under a 254 nm UV lamp. When developed using a modified potassium bismuth iodide reagent, orange-red spots were observed. f The value is 0.33. HRESIMS shows that its quasi-molecular ion [M+H] + Peak m / z 249.1960 (calculated value 249.1961), the molecular formula is deduced to be C 15 H 24 N2O, with an unsaturation degree of 5. This compound is similar in structure to the compound scutellarine A. The main difference in 1D NMR data is the absence of double bonds at the C-13 and C-14 positions. Its skeleton is also a matrine-type alkaloid. 1 H- 1This was also confirmed by H COSY and HMBC spectra. Correlated signals from H-17b / H-6, H-7, and H-11; H-2b / H-6; and H-2a / H-5 in the NOESY spectrum indicate that H-11, H-7, and H-6 are in the β-configuration, while H-5 is in the α-configuration, thus confirming the relative configuration of the compound. Two absolute configurations are possible. Using the same method as for scutellarine A, ECD spectra of the two enantiomers, 5S, 6R, 7S, 11R and 5R, 6S, 7R, 11S, were calculated. The results showed that the ECD curve for the 5S, 6R, 7S, 11R configuration closely matched the experimentally measured ECD curve, confirming the absolute configuration of the compound to be 5S, 6R, 7S, 11R. This compound was named scutellarine B.

[0265] The NMR spectrum data of scutellarine A and scutellarine B are shown in the following table:

[0266] Table 2. 1 H (600MHz) and 13 C(150MHz) data (CD3OD)

[0267] (5) Spectral data of celery scutellarin I

[0268] 1 H NMR (600MHz, CD3OD): δ8.04 (d, J=9.0Hz, 2H, H-2′, 6′), 6.89 (d, J=9.0Hz, 2H, H-3′, 5′),6.37(d,J=2.1Hz,1H,H-8),6.17(d,J=2.1Hz,1H,H-6),5.73(d,J=7.6Hz,1H,H- G1),5.24(d,1.4Hz,1H,H-R1),4.05(dd,J=9.6,6.2Hz,1H,H-R5),4.01(dd,J=3.4, 1.4Hz,1H,H-R2),3.79(dd,J=9.6,3.4Hz,1H,H-R3),3.73(dd,J=12.1,2.4Hz,1H,HG 6a ),3.62(dd,J=9.2,7.6Hz,1H,H-G2),3.57(dd,J=9.3,7.6Hz,1H,H-G3),3.51(dd,J=12.1,5.6Hz,1H,HG 6b ),3.35(t,J=9.6Hz,1H,H-R4),3.29(m,1H,H-G4),3.24(m,1H,H-G5),0.97(d,J=6.2Hz,3H,H3-R6).13 C NMR (150MHz, CD3OD): δ179.4(C-4),165.6(C-7),163.1(C-5),161.2(C-2),158.6(C-4′),1 58.4(C-9),134.5(C-3),132.1(C-2′,6′),123.2(C-1′),116.1(C-3′,5′),106.0(C-10),1 02.6(C-R1),100.3(C-G1),99.7(C-6),94.6(C-8),80.0(C-G2),78.9(C-G3),78.2(C-G5), 74.1(C-R4),72.4(C-R2),72.3(C-R3),71.9(C-G4),69.9(C-R5),62.7(C-G6),17.5(C-R6).

[0269] (6) Spectral data of celery quinone II

[0270] 1 H NMR (600MHz, CD3OD): δ8.05 (d, J=8.9Hz, 2H, H-2′, 6′), 6.87 (d, J=8.9Hz, 2H, H-3′, 5′), 6.39 (d, J=1. 6Hz,1H,H-8),6.20(d,J=1.6Hz,1H,H-6),5.25(d,J=7.5Hz,1H,H-G1),3.69(dd,J=11.9,2.5Hz,1H,HG 6a ),3.53(dd,J=11.9,5.5,Hz,1H,H-G2),3.41(m,2H,H-G3,G 6b ),3.30(m,1H,H-G5),3.20(m,1H,H-G4). 13 C NMR (150MHz, CD3OD): δ179.5(C-4),166.5(C-7),163.0(C-5),161.6(C-4′),159.1(C-2),158.6(C-9),135.5(C-3),132.3(C-2′,6′),122.9(C -1′),116.1(C-3′,5′),105.6(C-10),104.3(C-6),100.1(C-G1),94.9( C-8),78.4(C-G2),78.1(C-G5),75.8(C-G3),71.4(C-G4),62.7(C-G6).

[0271] (7) Spectral data of celery Ⅲ

[0272] 1 H NMR (600MHz, CD3OD): δ8.10(d,J=9.0Hz,2H,H-2′,6′),6.93(d,J=9.0Hz,2H,H-3′,5′),6.41(d,J=2.1Hz,1H,H-8),6.21(d,J=2.1Hz,1H,H-6). 13 C NMR (150MHz, CD3OD): δ177.4(C-4), 165.6(C-5), 162.5(C-7), 160.5(C-4′), 158.3(C-9), 148.1(C-2), 137.1 (C-3), 130.7 (C-2′, 6′), 123.8 (C-1′), 116.3 (C-3′, 5′), 104.6 (C-10), 99.3 (C-6), 94.5 (C-8).

[0273] Example 3:

[0274] The pharmaceutical composition of the present application is prepared by uniformly mixing 66.0 g of a scutellarin component and 3.0 g of a matrine component. The scutellarin component includes 27.0 g of scutellarin I, 33.0 g of scutellarin II, 5.0 g of scutellarin III, 0.5 g of scutellarin A, and 0.5 g of scutellarin B; and the matrine component includes 1.5 g of scutellarin A and 1.5 g of scutellarin B. Scutellarin A, scutellarin B, scutellarin A, and scutellarin B are all prepared as in Example 1.

[0275] Example 4:

[0276] The pharmaceutical composition of the present application is uniformly mixed with 60.0 g of a scutellarin component and 5.0 g of a matrine component. The scutellarin component includes 30.0 g of scutellarin A and 30.0 g of scutellarin B; and the matrine component includes 2.5 g of scutellarin A and 2.5 g of scutellarin B. Scutellarin A, scutellarin B, scutellarin A, and scutellarin B are all prepared as in Example 1.

[0277] Example 5:

[0278] The pharmaceutical composition of the present application is prepared by uniformly mixing 65.0 g of a scutellarin component and 5.0 g of a matrine component. The scutellarin component includes 27.0 g of scutellarin I, 33.0 g of scutellarin II, and 5.0 g of scutellarin III; and the matrine component includes 2.0 g of matrine, 2.0 g of sophocarpine, 0.5 g of scutellarine A, and 0.5 g of scutellarine B. Both scutellarine A and scutellarine B are prepared as in Example 1.

[0279] Example 6:

[0280] The pharmaceutical composition of the present application is prepared by uniformly mixing 60.0 g of celastrol components and 20.0 g of matrine components, wherein the celastrol components include 30.0 g of celastrol I and 30.0 g of celastrol II; and the matrine components include 10.0 g of matrine and 10.0 g of sophocarpine.

[0281] Example 7:

[0282] The pharmaceutical composition of the present application is prepared by uniformly mixing 60.0 g of celastrol components and 60.0 g of matrine components, wherein the celastrol components include 30.0 g of celastrol I and 30.0 g of celastrol II; and the matrine components include 30.0 g of matrine and 30.0 g of sophoracarpine.

[0283] Example 8:

[0284] The pharmaceutical composition of the present application is prepared by uniformly mixing 60.0 g of celastrol components and 5.0 g of matrine components, wherein the celastrol components include 60.0 g of celastrol III; and the matrine components include 5.0 g of matrine.

[0285] Performance testing:

[0286] Evaluation of anti-inflammatory effects of different drug combinations

[0287] (1) Experimental methods

[0288] RAW 264.7 cells (2×10 4 ) cells were seeded into a 96-well plate, and the original culture medium was removed after the cells adhered to the wall. 200 μL of DMEM culture medium was added to the control group, 200 μL of DMEM culture medium containing LPS (1 μg / mL) was added to the model group, and 200 μL of DMEM culture medium containing LPS (1 μg / mL) and the test drug were added to the drug-treated group. After incubation for 24 hours, 100 μL of culture supernatant was removed from each well and placed in another 96-well plate. Then, 100 μL of Griess reagent was added to each well. After 15 minutes, the absorbance of the solution was measured at 570 nm. The concentration of NO can be calculated based on the NaNO2 standard curve.

[0289] The MTT assay was performed in a standard 96-well plate. 20 μL of MTT solution (2 mg / mL) was added to each well. After a 3-hour incubation, the culture supernatant was aspirated and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. After 5 minutes, cell viability was measured by measuring absorbance at 570 nm.

[0290] (2) Experimental results

[0291] Each pharmaceutical composition had no cytotoxicity within the dose range of 2-32 μg / mL and was able to inhibit the production of NO in a dose-dependent manner. At a dose of 32 μg / mL, the maximum inhibition rates were 79.02% (composition of Example 3, Figure 10A), 89.39% (composition of Example 4, Figure 10B), 77.02% (composition of Example 5, Figure 10C), 63.86% (composition of Example 6, Figure 10D), 51.49% (composition of Example 7, Figure 10E) and 86.88% (composition of Example 8, Figure 10F), respectively. The compositions of Example 4 and Example 8 showed better anti-inflammatory effects.

[0292] Inhibitory effect of scutellariae fractions on nitric oxide (NO) production

[0293] (1) Experimental methods

[0294] RAW 264.7 cells (2×10 4 ) cells were seeded into a 96-well plate, and the original culture medium was removed after the cells adhered to the wall. 200 μL of DMEM culture medium was added to the control group, 200 μL of DMEM culture medium containing LPS (1 μg / mL) was added to the model group, and 200 μL of DMEM culture medium containing LPS (1 μg / mL) and the test drug were added to the drug-treated group. After incubation for 24 hours, 100 μL of culture supernatant was removed from each well and placed in another 96-well plate. Then, 100 μL of Griess reagent was added to each well. After 15 minutes, the absorbance of the solution was measured at 570 nm. The concentration of NO can be calculated based on the NaNO2 standard curve.

[0295] The MTT assay was performed in a standard 96-well plate. 20 μL of MTT solution (2 mg / mL) was added to each well. After a 3-hour incubation, the culture supernatant was aspirated and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. After 5 minutes, cell viability was measured by measuring absorbance at 570 nm.

[0296] (2) Experimental results

[0297] Compounds celecoxib III, celecoxib A and celecoxib B were non-cytotoxic within the dose range of 3.13-100 μM and were able to inhibit the production of NO in a dose-dependent manner. At a dose of 100 μM, the maximum inhibition rates were 85.46% (celecoxib III), 59.48% (celecoxib A) and 60.59% (celecoxib B), respectively ( Figure 11 ).

[0298] Quinone reductase (QR) induction activity of scutellarin components

[0299] Experimental methods

[0300] Hepa1c1c7 cells (2×10 4 ) cells were seeded into a 96-well plate, and the original culture medium was removed after the cells adhered to the wall. 200 μL of DMEM medium was added to the control group, and 200 μL of DMEM medium containing different concentrations of the test drug was added to the treatment group. After 24 hours of incubation, the original culture medium was removed, and 30 μL of cell lysis buffer was added to each well. After incubation for 15 minutes, 170 μL of the complete reaction mixture was added to each well. After 4 minutes, the absorbance of the solution was measured at 630 nm.

[0301] Experimental results

[0302] Compounds celecoxib III, celecoxib A and celecoxib B can induce QR activity in a dose-dependent manner. At a dose of 100 μM, the induction fold of celecoxib III can reach 3.65; at a dose of 100 μM, the maximum induction folds of celecoxib A and celecoxib B are 3.65 and 2.13, respectively ( Figure 12 ).

[0303] Effects of scutellarin and matrine components on the transcriptome of human bronchial epithelial Beas-2B cells

[0304] (1) Experimental methods

[0305] A Beas-2B cell inflammation model was induced with LPS and divided into a blank group, an inflammation group, a scutellarin A group, and a scutellarin A group. Transcriptomic analysis was performed using an Illumina NovaSeq6000 sequencer in the PE150 sequencing mode. After obtaining raw data, differentially expressed genes were analyzed using the R language.

[0306] (2) Experimental results

[0307] Using |logFC|>1 as the screening condition, compared with the blank group, the inflammation group had 1782 upregulated genes and 875 downregulated genes (Figure 13A, Figure 14A). Compared with the inflammation group, the clematisin A group had 303 upregulated genes and 76 downregulated genes (Figure 13B); the clematisin A group had 243 upregulated genes and 92 downregulated genes (Figure 14B). Using the website Venny2.1.0, the upregulated differential genes in the inflammation group and the downregulated differential genes in the drug-added group, as well as the downregulated differential genes in the inflammation group and the upregulated differential genes in the drug-added group were intersected, and 379 potential anti-inflammatory functional genes (targets) for clematisin A and 335 potential anti-inflammatory functional genes (targets) for clematisin A and 335 potential anti-inflammatory functional genes (targets) for clematisin A were initially obtained. The intersection of the two was only 42 (Figure 15), indicating that there are significant differences in the inflammatory regulatory genes of the two components and that the anti-inflammatory mechanisms of the two components are different.

[0308] The effectiveness of scutellarin and matrine components in the intervention of pulmonary inflammatory diseases and their synergistic mechanism

[0309] This study demonstrated the significant therapeutic effects of scutellarin and matrine compounds on lung inflammation. In-depth investigation of the underlying mechanisms revealed that scutellarin and matrine compounds inhibited inflammatory responses and oxidative stress by suppressing increases in NLRP3, caspase-1, IL-1β, and COX-2 mRNA levels. A novel mechanism for scutellarin in treating acute lung injury was identified, which involved regulating ferroptosis-related pathway proteins SLC7A11 and GPX4. Matrine compounds inhibited overactivation of the NLRP3 inflammasome and NF-κB activation. Furthermore, scutellarin and matrine compounds showed regulatory effects on distinct intestinal microbiota. Scutellarin compounds primarily increased the abundance of murinus in Lactobacillus, while matrine compounds primarily increased the abundance of caecimuris and vulgatus in Bacteroides. Correlations between these bacterial strain changes and the expression of immune factors were also observed. The two work synergistically to inhibit inflammatory responses and oxidative stress, thereby inhibiting the progression of inflammatory lung diseases.

[0310] 1. The effectiveness of scutellarin and matrine components in the intervention of acute lung injury and their synergistic mechanism

[0311] (1) Experimental methods

[0312] C57BL / 6N male mice (6-7 weeks old, 19-21 g) (purchased from Beijing Weitonglihua Company) were placed in an animal room with constant temperature and humidity under a 12-h dark / light cycle and had free access to water and food.

[0313] Animal grouping: 50 mice were randomly divided into 10 groups (n=5), namely LPS (10 mg / kg) group, control group (CTRL), positive control drug (DEX, 1 mg / kg) group, basilixin I (BS1, 20 mg / kg) group, basilixin II (BS2, 20 mg / kg) group, basilixin III (BS3, 20 mg / kg) group, matrine (MR, 20 mg / kg) group, and combination (BS3+MR, basilixin III 20 mg / kg + matrine 20 mg / kg) group.

[0314] Modeling and Dosing: 10 mg / kg of LPS (dissolved in saline) was instilled into the trachea of ​​mice using a liquid aerosol pulmonary drug delivery device. Drugs were administered orally 4 hours before modeling. All drugs were dissolved in 5% ethanol and saline. Dosing and modeling were repeated every other day. Mice were sacrificed on the 14th day.

[0315] Complete blood cell count: 24 hours after the last dose of the drug, peripheral blood was obtained by removing the eyeballs of the mice and placed in 5 mL EDTAK2 anticoagulant tubes. Complete blood cell count was performed using a Mindray BC-6800 fully automated hematology analyzer.

[0316] Bronchoalveolar lavage fluid (BALF) collection and ELISA analysis: Mice were sacrificed by cervical dislocation and dissected to expose the trachea. 1 mL of PBS was injected into the trachea using a syringe. After 45 seconds, the BALF was collected and repeated three times. The BALF was centrifuged at 2000 rpm for 10 minutes. The supernatant was the BALF and stored at -20°C. BALF was analyzed by enzyme-linked immunosorbent assay (ELISA).

[0317] Histopathological evaluation: Freshly removed right lower lobes of mouse lungs were fixed in 4% paraformaldehyde for 24 hours, dehydrated, and then embedded in paraffin. Mouse lung tissue was cut into 4 μm sections and dewaxed; then washed three times with xylene I (16 minutes); xylene II (16 minutes); anhydrous ethanol I (6 minutes); anhydrous ethanol II (6 minutes); 75% alcohol (6 minutes); and water. After dewaxing, sections were stained with hematoxylin and eosin (H&E).

[0318] Lung tissue homogenate protein assay: Tissue proteins were collected, denatured, and ultrasonically disrupted. After centrifugation, the cell lysate supernatant was analyzed by SDS gel electrophoresis, transferred to a membrane, blocked, and then the corresponding antibody was added. The cells were incubated overnight at 4°C, washed with PBST, and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour for protein analysis.

[0319] The qRT-PCR method was used to detect the levels of inflammatory factor mRNA: the mouse lung tissue was minced and total mRNA was extracted using Trizol reagent. After reverse transcription, the mRNA levels of NLRP3, caspase-1, IL-1β, COX-2, SLC7A11, and GPX4 were detected by qRT-PCR.

[0320] Statistical analysis: Graph Pad Prism 8.0 software was used for analysis. One-way analysis of variance (ANOVA) and LSD test were used to compare the differences between the groups. Significant differences were observed when P < 0.05 or P < 0.01. The results are expressed as mean ± SD.

[0321] (2) Experimental results

[0322] Both basidiosid and matrine components can attenuate LPS-induced lung pathological damage in mice. The lung tissue structure of the control group was intact, without hemorrhage and edema. In contrast, the LPS group showed continuous infiltration of inflammatory cells, accumulation of neutrophils in the alveoli and interstitial spaces, thickening of the alveolar and airway walls, and severe hemorrhage. The basidiosid I (BS1), basidiosid II (BS2), basidiosid III (BS3), matrine (MR), and combination treatment groups all significantly alleviated these inflammatory symptoms, with the combination group showing superior effects (Figure 16).

[0323] Both basilixin components and matrine components can inhibit the production of LPS-induced inflammatory factor IL-1β in the lungs of mice. The level of IL-1β in the alveolar lavage fluid of mice in the LPS model group was significantly increased. The addition of basilixin I (BS1), basilixin II (BS2), basilixin III (BS3), matrine (MR) and the combination reduced the level of inflammatory factor IL-1β, and the combined administration of basilixin III and matrine was better than that of either alone (Figure 17).

[0324] Both basilicin and matrine components reduced the number of inflammatory cells in the peripheral blood of mice induced by LPS. The number of white blood cells and neutrophils in the blood of mice in the LPS model group increased significantly, indicating that LPS caused systemic inflammation in the mice. Treatment with basilicin I (BS1), basilicin II (BS2), basilicin III (BS3), matrine (MR), and the combination reduced the number of both. Therefore, both basilicin and matrine components can reduce systemic inflammatory responses (Figure 18).

[0325] The basidiosid components and matrine components inhibited the LPS-induced increase in the mRNA levels of inflammatory factors in the lung tissue of mice. qRT-PCR results showed that basidiosid I (BS1), basidiosid II (BS2), basidiosid III (BS3), matrine (MR) and the combination were able to inhibit the increase in the mRNA levels of NLRP3, caspase-1, IL-1β and COX-2 ( Figure 19 ).

[0326] Components of the basidiol class can regulate ferroptosis-related pathways to exert lung protection. The ferroptosis-related pathway proteins SLC7A11 and GPX4 in the LPS model group were significantly reduced. The administration of basidiol I (BS1), basidiol II (BS2), and basidiol III (BS3) significantly promoted the increase of SLC7A11 and GPX4 (Figure 20).

[0327] 2. The effectiveness of scutellarin and matrine components in the intervention of chronic obstructive pulmonary disease and their synergistic mechanism

[0328] (1) Experimental methods

[0329] C57BL / 6N male mice (6-7 weeks old, 19-21 g) (purchased from Beijing Weitonglihua Company) were placed in an animal room with constant temperature and humidity under a 12-h dark / light cycle and had free access to water and food.

[0330] Animal grouping: 25 mice were randomly divided into 5 groups (n=5), namely, control (CTRL) group, cigarette smoke induced (CS) group, positive control drug (DEX, 1 mg / kg) group, cleistoglossine component (cleistoglossine A, BSA, 20 mg / kg) group, and matrine component (cleistoglossine A, BJA, 20 mg / kg) group.

[0331] Modeling and Dosing: A COPD model was established using cigarette smoke. Mice were exposed to cigarette smoke daily for 16 weeks. Drug treatment was administered at week 8. Drugs were administered 4 hours before induction. Drugs were dissolved in 5% ethanol and saline. Cigarette smoke was administered for 2 hours daily, 6 days per week, and drug treatment was administered once daily.

[0332] Complete blood cell count: 24 hours after the last dose of the drug, peripheral blood was obtained by removing the eyeballs of the mice and placed in 5 mL EDTAK2 anticoagulant tubes. Complete blood cell count was performed using a Mindray BC-6800 fully automated hematology analyzer.

[0333] Bronchoalveolar lavage fluid (BALF) collection and ELISA analysis: Mice were sacrificed by cervical dislocation and dissected to expose the trachea. 1 mL of PBS was injected into the trachea using a syringe. After 45 seconds, the BALF was collected and repeated three times. The BALF was centrifuged at 2000 rpm for 10 minutes. The supernatant was the BALF and stored at -20°C. BALF was analyzed by enzyme-linked immunosorbent assay (ELISA).

[0334] Histopathological evaluation: Freshly removed right lower lobes of mouse lungs were fixed in 4% paraformaldehyde for 24 hours, dehydrated, and then embedded in paraffin. Mouse lung tissue was cut into 4 μm sections and dewaxed; then washed three times with xylene I (16 minutes); xylene II (16 minutes); anhydrous ethanol I (6 minutes); anhydrous ethanol II (6 minutes); 75% alcohol (6 minutes); and water. After dewaxing, sections were stained with hematoxylin and eosin (H&E).

[0335] Detection of lung tissue lymphocyte subtypes: Mouse lung tissue was minced, digested with collagenase, ground and filtered, separated by PERCOLL gradient, lysed red blood cells, centrifuged and resuspended, membrane antibody stained, stimulated for differentiation, membrane permeabilization, intracellular antibody staining, centrifuged and resuspended, and tested on the machine.

[0336] Mouse fecal 16srRNA detection: Collect 3-4 pieces of mouse feces in a sterile EP tube, perform DNA extraction and purification, PCR amplification, product purification, library preparation and library testing, and NovaSeq detection.

[0337] Statistical analysis: Graph Pad Prism 8.0 software was used for analysis. One-way analysis of variance (ANOVA) and LSD test were used to compare the differences between the groups. Significant differences were observed when P < 0.05 or P < 0.01. The results are expressed as mean ± SD.

[0338] (2) Experimental results

[0339] Both scutellarin and matrine fractions attenuated CS-induced lung pathological damage in mice. The control group showed intact lung tissue structure, without hemorrhage or edema. In contrast, the CS group exhibited enlarged alveolar domains and increased alveolar tissue damage. Both scutellarin and matrine fractions alleviated these disease symptoms (Figure 21).

[0340] Both celastrol and matrine components can inhibit the production of inflammatory factors IL-1β and TNF-α in the lungs of mice induced by CS. The levels of IL-1β and TNF-α in the alveolar lavage fluid of mice in the CS model group were significantly increased. Treatment with celastrol and matrine components reduced the levels of inflammatory factors IL-1β and TNF-α (Figure 22).

[0341] Both scutellarin and matrine components reduced the number of inflammatory cells in the peripheral blood of mice induced by CS. The number of white blood cells and neutrophils in the blood of mice in the CS model group increased significantly, indicating that CS caused systemic inflammation in the mice. Treatment with scutellarin and matrine components reduced the number of both. Therefore, both scutellarin and matrine components can reduce systemic inflammatory responses (Figure 23).

[0342] The scutellarin and matrine compounds inhibited the ratio of Th1 and Th17 cells in the lung tissues of CS-induced mice. In CS mice, the ratio of Th1 and Th17 cells increased significantly, disrupting the balance between Th1 and Th2 cells and between Th17 and Treg cells, thereby triggering an immune response. The results showed that the scutellarin and matrine compounds significantly inhibited the ratio of Th1 and Th17 cells, thereby normalizing the balance between Th1 and Th2 cells and between Th17 and Treg cells (Figures 24 and 25).

[0343] The chelidonin components and matrine components can improve the composition of the intestinal flora of CS-induced COPD mice. In CS-induced mice, the intestinal flora was imbalanced compared with the control group mice. After intervention with chelidonin components and matrine components, the composition of the intestinal flora was significantly improved. Among them, the chelidonin components mainly improved the abundance of murinus in Lactobacillus, while the matrine components mainly improved the abundance of caecimuris and vulgatus in Bacteroides to improve COPD (Figure 26).

[0344] Binding of scutellarin and matrine components to key inflammatory regulatory proteins

[0345] (1) Experimental methods

[0346] The structure of the compound was established using ChemDraw 20.0 software and converted into a three-dimensional structure using Chem3D. The structural coordinates of INOS and COX-2 were retrieved from the RCSB Protein Data Bank (www.rcsb.org; PDB ID: 3E6T; 5IKQ) in PDB format. Molecular docking was performed using the Maestro 12.8 module within the software. The protein preparation wizard preprocessed and optimized the protein structure. Subsequently, molecular docking studies between ligands and proteins were performed using Glide using the standard precision (SP) docking mode and default parameters. Binding interactions were analyzed. Ligands and target proteins were ranked according to their Glide scores.

[0347] (2) Experimental results

[0348] Representative scutellarin and matrine compounds showed strong binding to the key inflammatory regulatory proteins iNOS and COX-2. Among them, the new scutellarin compounds scutellarin A and B had much better binding to iNOS and COX-2 than the known scutellarin compound scutellarin III. The new matrine compound scutellarin B also had better binding to iNOS than the known matrine compounds matrine and sophocarpine.

[0349] The binding affinity of representative scutellarin and matrine components to key inflammatory regulatory proteins is shown in Table 3 below:

[0350] Table 3 Binding affinity of representative scutellarin and matrine components to key inflammatory regulatory proteins

[0351] The scutellarin-like components inhibit the hepatotoxicity of matrine-like components

[0352] 1. Combination of celery compounds and matrine reduces the hepatotoxicity of matrine

[0353] (1) Experimental methods

[0354] Human liver immortalized THLE-2 cells (2×10 4 Each well was inoculated with 100 μL of MTT solution (2 mg / mL) and incubated overnight. The original culture medium was aspirated and culture medium containing the test concentration of matrine or matrine and scutellarin was added and incubated for 24 hours. The original culture medium was discarded and 20 μL of MTT solution (2 mg / mL) was added to each well. After incubation for 3 hours, the culture supernatant was aspirated and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. After 5 minutes, cell viability was measured by measuring the absorbance at 570 nm.

[0355] (2) Experimental results

[0356] Matrine (MR) is toxic to normal liver cells, inhibiting cell viability in a dose-dependent manner with a 50% inhibitory concentration of 14.1 μM. Combination with basidioscin A (BSA) and basidioscin III (BS3) effectively reduced matrine's nephrotoxicity. The 50% inhibitory concentration of MR increased to 35.5 μM when combined with BSA and 46.8 μM when combined with BS3 (Figure 27).

[0357] 2. Combination of celery compounds and matrine reduces matrine-induced reactive oxygen species generation

[0358] (1) Experimental methods

[0359] Human liver THLE-2 cells (2×10 5 / well) were inoculated in D35 and incubated overnight. The original culture medium was aspirated, and culture medium containing the test concentration of matrine or matrine and scutellarin components was added and incubated for 24 hours. The original culture medium was discarded, and the cells were collected after digestion with EDTA-free trypsin and washed twice with PBS. 200 μL DCHF-DA (200 μg / mL) solution was added and incubated for 2 hours. The cells were washed three times with PBS, and the cellular ROS levels were detected by flow cytometry.

[0360] (2) Experimental results

[0361] Matrine (MR) can induce oxidative stress in THLE-2 cells and dose-dependently increase ROS levels in liver cells, with a maximum induction fold of 2.37. Combination with basidiomycoside A (BSA) and basidiomycoside III (BS3) can effectively reduce ROS levels in response to matrine. The maximum induction fold of matrine in combination with BSA was reduced to 1.63-fold, and the maximum induction fold of matrine in combination with BS3 was reduced to 1.82-fold (Figure 28).

[0362] Matrine components improve the water solubility of celastrol components

[0363] (1) Experimental methods

[0364] Weigh an excess of kaempferol (kaempferol III), add 1.0 mL of distilled water to the blank group and 1.0 mL of matrine aqueous solution to the mixed group, and mix well. Place in an ultrasonic cleaner and sonicate for 30 minutes at 8000 r / min. -1 The mixture was centrifuged for 10 min (centrifugal radius 12 cm), filtered through a 0.22 μm microporous filter membrane, and analyzed by HPLC. The peak area was recorded. The peak area of ​​kaempferol in the blank group was A1, and the peak area of ​​kaempferol in the mixed group was A2. The peak area increase was calculated according to the formula: peak area increase = (A2 - A1) / A1.

[0365] (2) Experimental results

[0366] The peak area of ​​kaempferol in the mixed group increased by 7 times compared with that in the blank group. The matrine components can greatly improve the water solubility of the celery extract components.

[0367] In summary, the pharmaceutical composition provided in the present application mainly includes celastrol components and matrine components. The celastrol components have QR inducing activity, can inhibit the excessive production of NO in RAW264.7 mouse macrophages induced by LPS, reduce LPS-induced pathological changes in mouse lung tissue, inhibit the increase of inflammatory factor IL-1β in the alveolar lavage fluid of mice induced by LPS, and can reduce the number of inflammatory cells in the blood of mice, thereby exerting a lung protective effect and alleviating lung inflammation. The synergistic and enhancing effects of chelidonin components and matrine components, the two of which play a role in treating lung diseases through different mechanisms of action. Among them, chelidonin components have a new mechanism for treating acute lung injury by regulating ferroptosis-related pathway proteins SLC7A11 and GPX4. At the same time, matrine components can inhibit the excessive activation of NLRP3 inflammasome and inhibit NF-kB activation; the two synergistically exert lung protection effects through different mechanisms of action; and the binding of chelidonin and matrine components to key inflammatory regulatory proteins has better anti-inflammatory effects. Therefore, the two components have an interaction of "enhancing efficacy and reducing toxicity, complementary mechanisms, and mutual solubility", which is conducive to wide application.

[0368] The above are merely optional embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included within the scope of the claims of the present application.

Claims

1. A pharmaceutical composition for treating pulmonary inflammatory diseases, characterized in that, It comprises the following components: The compositae of the herba thesii components 1-100 parts, The matrine components 1-20 parts; Among them, the general structural formula of the thesium chinense components is as follows: Wherein, R4 is H or a group; R1, R2, R3, R5 are independently selected from one of the following groups: H, CH3, CH3CO, glucosyl, rhamnosyl, glucorhamnosyl, glucoglucosyl, xylosyl, sucrose group; The general structural formula of the matrine components is as follows: Wherein, R6, R7, R8 are independently selected from one of the following groups: H, OH, OCH3, CH3COO.

2. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 1, wherein In the pharmaceutical composition, it comprises the following components: The compositae of the herba thesii components 60-66 parts, The matrine components 2-5 parts.

3. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 1, wherein, The compositae of the herba thesii components are selected from at least one of the compositae of the herba thesii I, the compositae of the herba thesii II, the compositae of the herba thesii III, the compositae of the herba thesii A, the compositae of the herba thesii B.

4. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 1, wherein The matrine components are selected from at least one of matrine, sophocarpine, thesion A, thesion B.

5. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 3 or 4, characterized in that, The pharmaceutical composition comprises the following components: The compositae of the herba thesii A 10-20 parts, The compositae of the herba thesii B 10-20 parts, Thesion A 1-2 parts, Thesion B 1-2 parts; Or, The pharmaceutical composition comprises the following components: The compositae of the herba thesii III 10-15 parts, Matrine 1 part.

6. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 3, wherein The extraction method of the compositae of the herba thesii A and the compositae of the herba thesii B comprises the following steps: After drying the whole herb of thesium chinense is crushed, it is soaked at 20-40 °C for 1-10 hours, and then heated under reflux with 60-80% ethanol To obtain an extract; the extract is filtered and concentrated in sequence to obtain a total extract; The total extract is suspended in water and extracted with petroleum ether, dichloromethane and ethyl acetate in sequence to obtain each organic extract; The ethyl acetate extract is separated by normal phase silica gel column chromatography. Among them, gradient elution is first carried out with an eluent of petroleum ether and ethyl acetate with a volume ratio of 0.1-1:0.1-1, and then gradient elution is carried out with an eluent of ethyl acetate / methanol with a volume ratio of 0.1-1:0.1-1; 8 parts are received in sequence according to the time order to obtain the first elution product E1-E8; After removing pigments from the E7 part of the first elution product through an MCI column, gradient elution is carried out with an eluent of methanol / water with a volume ratio of 1-3:0.1-7, and 8 parts are received in sequence according to the time order to obtain the second elution product E7a-h; The E7d part of the second elution product is separated by a positive silica gel column, and the eluent is gradient eluted with an eluent of dichloromethane / methanol with a volume ratio of 0.1-30:1, and 8 parts are received in sequence according to the time order to obtain the third elution product E7a-h; The E7d5 part of the third elution product is separated and purified by Sephadex LH-20 and C18 reverse phase silica gel to obtain the compounds compositae of the herba thesii A and compositae of the herba thesii B, Among them, the structural formula of the thesium chinense turczaninow A is as follows: The structural formula of the said thesium chinense turcz. saponin B is as follows:

7. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 4, wherein, The extraction method of thesion A and thesion B is: After pulverizing the whole herb of Thesium chinense Turcz., it was extracted with a hydrochloric acid aqueous solution with a mass percentage concentration of 0.1 - 0.5 mol / L. After adjusting the extract to alkaline, it was repeatedly extracted with dichloromethane, and the total alkaloid extract was obtained after concentration under reduced pressure; The total alkaloid extract was separated by a normal-phase silica gel column and eluted with an eluent of petroleum ether / dichloromethane / ammonia water with a volume ratio of 1:1:0.02 - 0.03; then gradient elution was carried out with an eluent of dichloromethane / methanol with a volume ratio of 6 - 97:3 - 4, and 10 fractions were sequentially received in time order to obtain the fourth elution products S1 - S10; The S5 fraction of the fourth elution product was purified by Sephadex LH-20 column chromatography with a methanol eluent, and the purified S5 fraction was separated by HPLC. Among them, elution was carried out with a mobile phase with a pH of 3 - 4 and a methanol volume percentage content of 5% - 10%; Impurities were removed by a macroporous adsorption resin column to obtain thesionine B and thesionine A; Among them, the structural formula of the thesion A is as follows: The structural formula of the thesion B is as follows:

8. The pharmaceutical composition for treating pulmonary inflammatory diseases according to claim 3, characterized in that, The extraction process of thesion I, thesion II, and thesion III includes the following steps: After pulverizing the dry whole herb of Thesium chinense Turcz., it was soaked at 20 - 40 °C for 1 - 10 hours, and then extracted by heating under reflux with an ethanol aqueous solution with a mass percentage content of 60 - 80%; after filtering the extract, the extract was concentrated to obtain the total extract; After suspending the total extract in water, it was successively extracted with petroleum ether, dichloromethane, and ethyl acetate to obtain each organic extract; After adding water to the ethyl acetate extract, it was passed through an MCI column, and then successively eluted with methanol-aqueous solutions with a mass percentage content of 20% - 30%, 40% - 50%, and 60% - 70% to obtain the first eluate, the second eluate, and the third eluate in sequence; The first eluate was successively eluted with pure water, methanol-aqueous solutions with a mass percentage content of 20% - 30%, 40% - 50%, 60% - 70%, and pure ethanol to obtain components a, b, c, d, and e in sequence; Among them, the b component was purified by a gel chromatography column with methanol as the eluent and recrystallized to obtain thesion I; The c component was purified by a gel chromatography column with methanol as the eluent and recrystallized to obtain thesion II; The third eluate was purified by a gel chromatography column with methanol as the eluent and recrystallized to obtain thesion III.

9. A preparation for a pulmonary inflammatory disease, characterized in that: The mass percentage of the pharmaceutical composition according to any one of claims 1 - 8 as an active component in the preparation is 0.1% - 99.9%, and the rest is a pharmaceutically acceptable excipient; among them, the dosage form of the preparation is tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, buccal tablets, granules, electuaries, pills, powders, plasters, pills, suspensions, powders, injections, suppositories, creams, sprays, drops, or patches.

10. Use of the pharmaceutical composition for treating pulmonary inflammatory diseases according to any one of claims 1 - 7 in the preparation of drugs for chronic obstructive pulmonary disease, acute lung injury, or lower respiratory tract infection.

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