Composition containing mixed herbal medicine extract as active ingredient for preventing or treating respiratory inflammatory diseases or sepsis

Abstract

The present invention relates to a composition containing a mixed herbal medicine extract as an active component for preventing or treating respiratory inflammatory diseases or sepsis.

Description

Composition for preventing or treating respiratory inflammatory disease or sepsis containing mixed herbal extract as an active ingredient

[0001] The present invention relates to a composition for preventing or treating respiratory inflammatory disease or sepsis, comprising a mixed herbal extract as an active ingredient.

[0002]

[0003] Respiratory inflammatory diseases (RIDs) are conditions that cause inflammation of the airways, bronchi, and lungs, often accompanied by symptoms such as coughing, phlegm, shortness of breath, and fever. Their mortality rate continues to rise due to increased exposure to air pollutants like fine dust and a growing smoking population. Known RIDs include acute lung injury, emphysema, asthma, chronic obstructive pulmonary disease (COPD), otitis media, sinusitis, tonsillitis, nasopharyngitis, laryngitis, and acute bronchitis.

[0004] Meanwhile, sepsis refers to a condition in which a systemic inflammatory response occurs, such as rapid body temperature changes, rapid heartbeat and respiratory rate, and pathological increases or decreases in white blood cell count, due to infection with microorganisms such as bacteria, fungi, parasites, and viruses. Sepsis is a disease that causes 6 million deaths worldwide each year. In Korea, the number of sepsis deaths has also been steadily increasing over the past decade, leading to a rapid increase in the resulting social burden.

[0005] Symptoms of sepsis vary depending on the type of infectious agent causing the disease, but the main cause of death is multiple organ damage caused by systemic inflammatory response syndrome. In particular, when acute lung damage occurs, it can lead to acute respiratory distress syndrome, negatively affecting survival rates.

[0006] Acute lung injury (ALI) is a life-threatening condition characterized by respiratory failure and lung inflammation. Acute lung injury can result from a variety of causes, including sepsis and pneumonia. Aseptic inflammation, such as trauma, burns, and shock, can also directly or indirectly cause ALI. During the progression of ALI, the barriers between the alveolar epithelium and pulmonary endothelium are disrupted, and the alveolar space, filled with protein-rich fluid and inflammatory cells, overflows. Furthermore, ALI is one of the most severe symptoms of COVID-19 infection, drawing increasing clinical and basic research attention.

[0007] Research and development using natural products is emerging as one of the treatment strategies for sepsis or acute lung injury, but research on this topic is still limited to date.

[0008]

[0009] The present inventors have made diligent efforts to develop a novel therapeutic agent that exhibits a therapeutic effect on respiratory inflammatory diseases or sepsis, and have completed the present invention by confirming that a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome exhibits a remarkably excellent therapeutic effect on respiratory inflammatory diseases or sepsis.

[0010]

[0011] One object of the present invention is to provide a pharmaceutical composition for preventing or treating respiratory inflammatory disease or sepsis, which comprises a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0012] Another object of the present invention is to provide a method for preventing or treating respiratory inflammatory disease or sepsis, comprising a step of administering the composition to a subject.

[0013] Another object of the present invention is to provide a food composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0014] Another object of the present invention is to provide a composition for preventing or treating respiratory inflammatory diseases or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0015] Another object of the present invention is to provide a feed composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0016] Another object of the present invention is to provide a use of a composition comprising a mixed extract of Astragali, Angelica, and Cnidium officinalis for the prevention or treatment of respiratory inflammatory diseases or sepsis.

[0017] Another object of the present invention is to provide a use of a composition comprising a mixed extract of Astragali, Angelica gigas, and Cnidium officinalis for the manufacture of a medicament for the prevention or treatment of respiratory inflammatory diseases or sepsis.

[0018]

[0019] The technical problem to be achieved according to the technical idea of ​​the invention disclosed in this specification is not limited to the problem to solve the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0020]

[0021] The mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome of the present invention exhibits a synergistic effect on respiratory inflammatory diseases or sepsis, and can be used as an excellent therapeutic agent.

[0022]

[0023] Figure 1 illustrates a Venn diagram of cross-targeting between a network of mixed extracts and a gene set of acute lung injury according to one embodiment of the present invention.

[0024] Figure 2 illustrates a network of mixed extracts and common genes of acute lung injury according to one embodiment of the present invention.

[0025] FIG. 3 is a COMPARTMENTS database analysis result for the potential functional mechanism of a mixed extract according to one embodiment of the present invention.

[0026] Figure 4 shows the enrichment analysis and detailed information extracted from the GO process of the target genes of the Osteo-F network.

[0027] Figure 5 shows the overlapping genes between GO:0,051,092, GO:0,043,122, and GO:0,071,347.

[0028] Figure 6 shows the results of overlapping gene analysis by GO biological process.

[0029] Figure 7 shows the top 20 genes overlapping between the mixed extract target genes and the netosis network according to one embodiment of the present invention.

[0030] FIG. 8 illustrates a Venn diagram of overlapping genes between mixed extracts and netosis according to one embodiment of the present invention.

[0031] Figure 9 shows common genes between mixed extracts and netosis according to one embodiment of the present invention.

[0032] FIG. 10 illustrates a network of mixed extracts and netosis according to one embodiment of the present invention, consisting of 12 nodes and 59 edges.

[0033] Figure 11 shows the results of confirming the survival rate after LPS injection to mice administered a mixed extract according to one embodiment of the present invention.

[0034] Figure 12 shows the results of confirming the survival time after LPS injection to mice administered a mixed extract according to one embodiment of the present invention.

[0035] Figure 13 shows the results of confirming the change in body weight after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0036] Figure 14 shows the results of H&E staining of lung sections after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0037] Figure 15 shows the results of confirming the lung damage score of lung sections after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0038] Figure 16 shows the results of confirming the wet / dry ratio of lung tissue after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0039] Figure 17 shows the results of H&E staining of BAL cells after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0040] Figure 18 shows the results of measuring the number of cells in BAL fluid, including PMNs, macrophages, and lymphocytes, after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0041] Figure 19 shows the results of confirming the decrease in cytokines in BAL solution after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0042] Figure 20 is a result of confirming the decrease in chemokines in BAL solution after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0043] Figure 21 shows the results of confirming cytokine suppression in plasma after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0044] Figure 22 shows the results of confirming the suppression of chemokines in plasma after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0045] Figure 23 shows the results of analyzing the composition of immune cells in the blood after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0046] Figure 24 shows the results of confirming the spleen size and spleen / BW ratio after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0047] Figure 25 shows the results of confirming Ly6G expression in PMNs isolated from bone marrow after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0048] Figure 26 shows the results of confirming the expression of CXCR2 and CXCR4 in PMNs isolated from whole blood after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0049] Figure 27 shows the results of confirming the regulation of NF-κB and MAPK pathways by a mixed extract according to one embodiment of the present invention.

[0050] Figure 28 shows the results of immunofluorescence staining of lung sections after LPS injection into mice administered a mixed extract according to one embodiment of the present invention.

[0051] Figure 29 shows the results of confirming the inhibition of inflammasome activity of a mixed extract according to one embodiment of the present invention.

[0052] Figures 30 and 31 show the results of confirming the inhibition of netosis formation by a mixed extract according to one embodiment of the present invention.

[0053] Figure 32 shows the results of confirming the inhibition of neutrophil extracellular trap formation in human PMN (hPMN) treated with a mixed extract according to one embodiment of the present invention.

[0054] Figure 33 shows the results of protein expression of NLRP3, MPO, and ELANE in hPMN treated with a mixed extract according to one embodiment of the present invention.

[0055] Figure 34 shows the results of confirming the elastase level in hPMN treated with a mixed extract according to one embodiment of the present invention.

[0056] Figure 35 shows the results of confirming the inhibition of ROS production in hPMN treated with a mixed extract according to one embodiment of the present invention.

[0057] Figure 36 shows a FlowSOM tree in hPMN treated with a mixed extract according to one embodiment of the present invention.

[0058] Figure 37 shows a tSNE projection in hPMN treated with a mixed extract according to one embodiment of the present invention.

[0059] Figure 38 shows a heatmap of analyzed PMN markers in hPMN treated with a mixed extract according to one embodiment of the present invention.

[0060] Figure 39 shows a tSNE projection of neutrophil extracellular trap-related markers and NLRP3 in hPMNs treated with a mixed extract according to one embodiment of the present invention.

[0061]

[0062] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0063] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. Furthermore, it should be understood that such equivalents are encompassed by the present invention.

[0064] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical field to which this application pertains and the content of this application.

[0065]

[0066] One aspect of the present invention provides a pharmaceutical composition for preventing or treating respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0067] Astragalus membranaceus is a perennial herb of the legume family in the Rosaceae order, native to Korea, Japan, Manchuria, northeastern China, and eastern Siberia. It is commonly cultivated as a medicinal herb. In oriental medicine, it is harvested in the fall, the outer layer and fine roots are removed, and the dried root is called astragalus membranaceus of herbal medicine. It is said to be more effective when used intact without peeling. It has the effects of strengthening, invigorating, diuretic, and reducing swelling, and is prescribed for physical weakness, fatigue, lethargy, and night sweats.

[0068] Angelica gigas is the dried root of Angelica gigas, a perennial herb belonging to the parsley family. It has a sweet and spicy taste and a warm nature. The efficacy of Angelica gigas is to replenish blood when there is a lack of blood.

[0069] It has a blood-replenishing effect that generates blood, and Angelica gigas promotes blood flow in the coronary arteries and stimulates red blood cell production. In addition, it contains decursin and / or decursinol angelate.

[0070] It has been reported that the extract of the ear can be used as an anticancer composition.

[0071] Trichosanthes kirilowii Maximowicz is the peeled root of the perennial hanultari or yellow hanultari, a member of the Cucurbitaceae family. It is odorless, bitter and sour in taste, and cool in nature. It is used to treat exhaustion, boils, and pus when body fluids are damaged by heat. It primarily reduces heat in the lungs and stomach, produces body fluids, quenches thirst, and moisturizes the body.

[0072] In the present invention, the Astragalus membranaceus, Angelica gigas Nakai, or Gastrodia elata extracts may be purchased commercially or may be collected or cultivated in nature, but are not limited thereto. Furthermore, the Astragalus membranaceus, Angelica gigas Nakai, or Gastrodia elata extracts may be extracted from natural, hybrid, or mutant plants, or may be extracted from plant tissue cultures.

[0073] In the present invention, the mixed extracts of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome are used interchangeably with "SH003" and have the same meaning.

[0074] In the present invention, the "mixed extract" may be a mixture of extracts obtained by extracting Astragalus membranaceus, Angelica gigas, and Gastrodia elata separately, or may be a mixture obtained by mixing Astragalus membranaceus, Angelica gigas, and Gastrodia elata and then extracting the mixture, but is not limited thereto.

[0075] For example, the "mixed extract" may be a mixture obtained by mixing extracts of all formulations that can be formed using the extracts, such as an extract obtained by extracting Astragalus membranaceus, Angelica gigas Nakai, and Gastrodia elata powder respectively, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a controlled or purified product of the extract, a fraction of the extract, or a mixture thereof. Alternatively, the mixed extract includes extracts of all formulations that can be formed using the extracts, such as an extract obtained by extracting a mixture of Astragalus membranaceus, Angelica gigas Nakai, and Gastrodia elata powder, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a controlled or purified product of the extract, a fraction of the extract, or a mixture thereof.

[0076] In one embodiment of the present invention, the extract may be obtained by extracting Astragalus membranaceus, Angelica gigas, and Cnidium rhizome with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, and may be obtained by extracting Astragalus membranaceus, Angelica gigas, and Cnidium rhizome with ethanol, but is not limited thereto.

[0077] In one embodiment of the present invention, the alcohol having 1 to 4 carbon atoms may be 10 to 90% (v / v) ethanol, and specific examples thereof may be 10 to 80% (v / v) ethanol, 10 to 70% (v / v) ethanol, 10 to 60% (v / v) ethanol, 20 to 60% (v / v) ethanol, 20 to 50% (v / v) ethanol, 20 to 40% (v / v) ethanol, and for example, 30% (v / v) ethanol, but is not limited thereto.

[0078] In the mixed extract of Astragalus membranaceus, Angelica gigas Nakai, and Cnidium officinalis of the present invention, the method for extracting the mixture is not particularly limited, and extraction can be performed according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed alone or in combination of two or more methods.

[0079] In addition, the above extract can be manufactured and used in the form of a dry powder after extraction, but

[0080] It is not limited anymore.

[0081] In one embodiment of the present invention, the mixed extract may be a weight ratio of Astragalus membranaceus: Angelica gigas Nakai: Gastrodia elata powder = 0.5~5 : 0.1~3 : 0.1~3, and for example, the weight ratio may be 1~5 : 0.5~2 : 0.5~2, 0.5~5 : 0.1~1.5 : 0.1~1.5, 1~4 : 0.5~1.5 : 0.5~1.5, 2~4 : 0.5~1.5 : 0.5~1.5 or 1~3 : 0.5~1 : 0.5~1, and in one specific example, the weight ratio may be mixed at 3 : 1 : 1, but is not limited thereto.

[0082] The term respiratory inflammatory disease of the present invention refers to a disease in which an inflammatory reaction occurs in the respiratory tract (nose, pharynx, larynx, bronchial tubes, etc.) or lung tissue, affecting respiratory function, and may appear in an acute or chronic form.

[0083] In the present invention, the respiratory inflammatory disease may be at least one selected from the group consisting of acute lung injury, emphysema, asthma, chronic obstructive pulmonary disease, otitis media, sinusitis, tonsillitis, nasopharyngitis, laryngitis, and acute bronchitis, but is not limited thereto.

[0084] In the present invention, the respiratory inflammatory disease may be caused by sepsis, but is not limited thereto.

[0085] The term "sepsis" in the present invention refers to a condition in which a systemic inflammatory response occurs, such as rapid changes in body temperature, increased tachycardia and respiratory rate, and pathological increases or decreases in white blood cell count, due to infection with microorganisms such as bacteria, fungi, parasites, and viruses. Sepsis can cause direct and serious inflammatory diseases in the respiratory tract, and can lead to various respiratory inflammatory diseases such as acute lung injury, pneumonia, bronchitis, lung abscess, or pleurisy.

[0086] In one embodiment of the present invention, the composition may suppress systemic inflammation.

[0087] In one embodiment of the present invention, the composition may suppress lung inflammation.

[0088] In one embodiment of the present invention, the composition may inhibit NF-κB and mitogen-activated protein kinase (MAPK) phosphorylation.

[0089] NF-κB, a transcription factor crucial for the immune response, is activated by proinflammatory stimuli and is involved in various human diseases. Inflammatory conditions can trigger NF-κB to induce the expression of iNOS, which increases NO production. NF-κB can also stimulate the expression of COX-2, enhancing the synthesis of prostaglandins and other inflammatory mediators. MAPKs include the ERK, JNK, and p38 pathways, which are associated with acute lung injury inflammation, and together with NF-κB, they regulate inflammasome activation.

[0090] In an embodiment of the present invention, it was confirmed that SH003 was treated in a mouse model in which acute lung injury was induced by administering LPS, thereby inhibiting phosphorylation of NF-κB and major MAPK family members, ERK, JNK, and p38 (Example 3-5).

[0091] In one embodiment of the present invention, the composition may inhibit inflammasome activation and neutrophil extracellular trap formation.

[0092] Inflammasomes serve as an essential mechanism in the innate immune system to control various inflammatory diseases by releasing IL-1β. Neutrophil extracellular traps (NETs) are formed by various stimuli, including pathogens, cytokines, and microcrystals. NETs are composed of DNA, histones (citrullinated), chromatin, and various enzyme proteins, such as MPO and neutrophil elastase (ELANE).

[0093] In an embodiment of the present invention, it was confirmed that SH003 was administered to a mouse model in which acute lung injury was induced by administering LPS, thereby reducing the expression level of inflammasome family proteins and inhibiting the formation of neutrophil extracellular traps (Examples 3-6 and 3-7).

[0094] The term "prevention" of the present invention means any act of inhibiting or delaying the onset of a respiratory inflammatory disease or sepsis by administering a pharmaceutical composition for preventing or treating a respiratory inflammatory disease or sepsis containing a mixed extract of Astragali, Angelica gigas, and Cnidium rhizome as an active ingredient.

[0095] The term "treatment" of the present invention means any action that improves or beneficially changes the symptoms of a subject suspected of having or developing respiratory inflammatory disease or sepsis by administering the pharmaceutical composition.

[0096] In the present invention, the composition is preferably used for humans, but may also be used for livestock such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs or cats that develop respiratory inflammatory diseases or sepsis and whose respiratory inflammatory diseases or sepsis can be suppressed or reduced by administration of the composition of the present invention.

[0097] The content of the mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium officinalis included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 80 wt%, 0.0001 to 50 wt%, or, for example, 0.01 to 20 wt%, based on the total weight of the final composition.

[0098] In the pharmaceutical composition of the present invention, in one specific example, the composition may additionally include, but is not limited to, a pharmaceutically acceptable carrier, excipient or diluent.

[0099] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may comprise a non-naturally occurring carrier.

[0100] More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, polycaprolactone, poly lactic acid, poly-L-lactic acid, mineral oil, and the like.

[0101] The term "pharmaceutically acceptable" in the present invention means that the composition exhibits properties of being non-toxic to cells or humans exposed to the composition.

[0102] Specifically, the pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, each according to a conventional method.

[0103] When formulating, they are usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0104] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives.

[0105] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0106] In the pharmaceutical composition of the present invention, in any one specific example, the composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, but is not limited thereto.

[0107] Another aspect of the present invention provides a method for preventing or treating respiratory inflammatory disease or sepsis, comprising administering the composition to a subject.

[0108] The definitions of the composition, respiratory inflammatory disease, sepsis, prevention and treatment are as described above.

[0109] Since the pharmaceutical composition of the present invention exhibits an effect of preventing or treating respiratory inflammatory diseases or sepsis, the method of the present invention including a step of administering the same to a subject can be usefully utilized for preventing or treating respiratory inflammatory diseases or sepsis.

[0110] The term "administration" used in the present invention means introducing the composition of the present invention into a subject by any appropriate method, and the route of administration of the composition may be through any common route as long as it can reach the target tissue.

[0111] For example, it may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, or intranasally, but is not limited thereto.

[0112] The composition of the present invention may be administered daily or intermittently, and the number of times per day it may be administered may be once or divided into 2 to 3 times.

[0113] The term "subject" in the present invention refers to any animal, including rats, mice, and livestock, including humans, that has developed or may develop respiratory inflammatory disease or sepsis. Specific examples include, but are not limited to, mammals, including humans.

[0114] The method for preventing or treating respiratory inflammatory disease or sepsis of the present invention may specifically include a step of administering to a non-human subject a pharmaceutically effective amount of a composition containing a mixed extract of Astragali, Angelica, and Cnidium officinalis.

[0115] The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be easily determined by those skilled in the art based on factors including the patient's sex, age, weight, health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0116] Specifically, the composition of the present invention can be administered at a daily dose of 0.0001 to 100 mg / kg of body weight, more specifically 0.001 to 100 mg / kg of body weight, based on solid content. The above recommended dosage can be administered once a day or divided into several doses.

[0117]

[0118] Another aspect of the present invention provides a food composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus Bunge, Angelica gigas Nakai, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0119] The above-mentioned Astragalus membranaceus, Angelica gigas, Cnidium japonica root, mixed extract, respiratory inflammatory disease and sepsis are as described above.

[0120] The term "improvement" of the present invention means any action that at least reduces the severity of a parameter, for example, a symptom, associated with a respiratory inflammatory disease and a septic condition by ingestion of the composition of the present invention.

[0121] When using the composition of the present invention as a health functional food additive, the composition can be added as is or combined with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredients mixed can be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Since the composition of the present invention has no stability issues, there are no significant restrictions on the amount mixed.

[0122] Since the food composition of the present invention can be consumed on a daily basis, it can be expected to have an effect in preventing, treating, or improving respiratory inflammatory diseases or sepsis, and thus can be very usefully used for health promotion purposes.

[0123] The term “food” in the present invention includes dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0124] The above term, "health functional food", is the same as food for special health use (FoSHU), and refers to a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. Here, "function" means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological action. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term administration of drugs because it uses food as a raw material, and is highly portable, so the health functional food of the present invention can be taken as a supplement to enhance the effect of improving respiratory inflammatory diseases or sepsis.

[0125] The above health functional foods refer to foods that have a more active health maintenance or promotion effect than regular foods, while health supplement foods refer to foods intended for health supplementation. In some cases, the terms health functional foods, health foods, and health supplements are used interchangeably.

[0126] Specifically, the health functional food means a food product manufactured by adding the composition of the present invention to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing it in the form of encapsulation, powder, suspension, etc., and means that when consumed, it brings about a specific health effect, but unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs by using food as a raw material.

[0127] The above food composition may additionally include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0128] In addition, the food composition may include additional ingredients commonly used in food compositions to improve odor, taste, appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and amino acids such as lysine, tryptophan, cysteine, and valine.

[0129] In addition, the food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), sterilizers (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar colorants, etc.), color developers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose fillers), film-forming agents, gum-forming agents, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0130] As an example, the food composition of the present invention can be used as a health beverage composition, and in this case, it can contain various flavoring agents or natural carbohydrates as additional ingredients like a regular beverage. The above-mentioned natural carbohydrates can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener can be a natural sweetener such as thaumatin and stevia extract; a synthetic sweetener such as saccharin and aspartame, etc. The proportion of the natural carbohydrate can be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 ml of the health beverage composition of the present invention.

[0131] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, the composition may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0132] The proportion of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0133]

[0134] Another aspect of the present invention provides a pharmaceutical composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus Bunge, Angelica gigas Nakai, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0135] The above-mentioned Astragalus membranaceus, Angelica gigas, Cnidium japonica root, mixed extract, respiratory inflammatory disease and sepsis are as described above.

[0136] The quasi-drug composition of the present invention may include, in addition to the mixed extract of Astragali, Angelica gigas, and Cnidium officinalis, ingredients commonly used in quasi-drug compositions, and may include, for example, an abrasive, a wetting agent, a binder, a foaming agent, a sweetener, a preservative, a medicinal ingredient, a flavoring agent, a coloring agent, a solvent, a whitening agent, a solubilizer, or a pH adjuster, but is not limited thereto.

[0137] In the present invention, the type of the above-mentioned quasi-drug is not particularly limited, and may be any quasi-drug commonly used in the relevant technical field. Non-limiting examples of the above-mentioned quasi-drug include one or more formulations selected from the group consisting of toothpaste, mouthwash, gum, candy, mouth spray, oral ointment, oral varnish, oral film, and gum massage cream. These may be used alone or in combination of two or more.

[0138]

[0139] Another aspect of the present invention provides a feed composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus Bunge, Angelica gigas Nakai, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

[0140] The above-mentioned Astragalus membranaceus, Angelica gigas, Cnidium japonica root, mixed extract, respiratory inflammatory disease and sepsis are as described above.

[0141] The term "feed" in the present invention means any natural or artificial diet, meal, etc. or ingredients of said meal for eating, ingesting, and digesting by an animal or suitable therefor.

[0142] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant feed such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal feed such as proteins, inorganic substances, oils, mineral substances, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0143] The mixed extract of the present invention included in the feed composition of the present invention may vary depending on the purpose of use and conditions of use of the feed, and for example, may be included in an amount of 0.01 to 100 wt%, more specifically 1 to 80 wt%, based on the total weight of the livestock feed composition.

[0144]

[0145] Another aspect of the present invention provides the use of a composition comprising a mixed extract of Astragali, Angelica, and Cnidium officinalis for the prevention or treatment of respiratory inflammatory diseases or sepsis.

[0146]

[0147] Another aspect of the present invention provides the use of a composition comprising a mixed extract of Astragali, Angelica, and Cnidium officinalis for the manufacture of a medicament for the prevention or treatment of respiratory inflammatory diseases or sepsis.

[0148]

[0149] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the embodiments according to the present invention may be modified in various ways, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0150]

[0151] Example 1: Preparation of a mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome

[0152] Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz were individually extracted with 30% ethanol for 3 hours and then freeze-dried according to the good manufacturing practice (GMP) procedure at Hanpung Pharmaceutical Co., Ltd. (Jeonju, Korea). The freeze-dried powders of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii were mixed in a weight ratio of 3:1:1 to prepare a mixture of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii (SH003). The mixture was diluted in phosphate-buffered saline (PBS) and stored at -80°C until further use.

[0153]

[0154] Example 2: In silico analysis of the association between mixed extracts of Astragali, Angelica, and Cnidium officinalis and acute lung injury.

[0155] 2-1. Construction of a network of mixed extracts of Astragali, Angelica, and Cnidium officinalis and comparison with the acute lung injury gene set.

[0156] The networks of Astragali, Angelica gigas Nakai, and Cnidium japonica were constructed using target genes of selected components of Astragali, Angelica gigas Nakai, and Cnidium japonica: formononetin, cucurbitacin D, and decurcin. Co-occurring target genes were collected from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). After combining genes and deleting duplicates, a total of 172 genes were identified, resulting in the construction of the Astragali, Angelica gigas Nakai, and Cnidium japonica networks. The entire networks of Astragali, Angelica gigas Nakai, and Cnidium japonica were truncated with a high confidence score of 0.7 or higher.

[0157] In addition, to compare the genes that cross-react between Astragali, Angelica, and Cnidium officinalis, a gene set of 'Acute lung injury (ALI)' was generated from the GeneCards database (http: / genecards.org / ). A total of 9,250 genes were identified to be related to acute lung injury, and the matching ratio was calculated.

[0158] As a result, the SH003 network, including Astragalus membranaceus, Cnidium japonica, and Angelica gigas, was confirmed to contain 172 nodes and 1,759 edges. To investigate the effect of SH003 on acute lung injury, the target genes of the SH003 network and the acute lung injury gene set were compared, and 152 intersecting genes were discovered (Figs. 1 and 2).

[0159] SH003 and acute lung injury showed an 88.4% concordance rate, indicating that the efficacy of SH003 is highly correlated with acute lung injury.

[0160]

[0161] 2-2. Predicting the potential functional mechanisms of mixed extracts of Astragali, Angelica, and Cnidium officinalis

[0162] Cytoscape functional enrichment analysis was performed using the SH003 network. Potential primary pathways and targets were sorted and classified by p-values ​​against the CMPARTMENTS database and the Gene Ontology (GO) biological process database.

[0163] Based on the significant p-value, the potential functional mechanisms of SH003 were found to be mainly related to 'NF-kappaB complex (GOCC:0071159)', 'NLRP3 inflammasome complex (GOCC:0072559)', and 'AIM2 inflammasome complex (GOCC:0097169)' in the COMPARTMENTS database (Fig. 3). 'Positive regulation of NF-kappaB transcription factor activity (GO:0051092)', 'Regulation of I-kappaB kinase / NF-kappaB signaling (GO:0043122)', 'Cellular response to interleukin-1 (GO:0071347)', 'Regulation of interleukin-1 beta production (GO:0032651)', and 'Regulation of interleukin-6 production (GO:0032675)' were identified as the underlying mechanisms of SH003 in the Biological Process Database (Fig. 4). GOCC:0071159 and GOCC:0072559 shared only IL1B, while GOCC:0072559 and GOCC:0097169 shared NLRP3, IL18, PYCARD, and CASP1 (Fig. 5). For GO biological processes, PYCARD, IL1B, STAT3, IL17A, TLR4, TNF, TRAF6, CASP1, ICAM1, RELA, and TNFSF11 appeared as overlapping genes (Fig. 6).

[0164]

[0165] 2-3. Network establishment between mixed extracts of Astragali, Angelica, and Cnidium japonica and neutrophil extracellular trap (NET) formation

[0166] We extracted 104 symbolic genes by searching the GeneCards database with the keyword NETosis and compared them with the SH003 target genes and the NETosis network.

[0167] A diagram image of "Neutrophil Extracellular Trap Formation - Homo sapiens (human)" was obtained from the 2020 Human Database of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The genes involved in SH003 are indicated in red boxes.

[0168] To explore the more specific mechanism of SH003, we compared the SH003 target gene and nepotosis networks. The top 20 genes that symbolize nepotism are listed in Figure 7. The total number of overlapping genes between SH003 and nepotism was 12 (Figure 8). In the SH003 target gene and nepotism gene set, 12 common genes, including AKT1, CXCL8, HIF1A, HMGB1, IL1B, IL6, IL17A, MMP2, MPO, NLRP3, PTEN, and TNF (Figure 9), showed a high correlation with nepotism, comprising 12 nodes and 59 edges (Figure 10).

[0169] Highlighting the target genes of SH003 in the KEGG pathway map 'Neutrophil extracellular trap formation (map04613)' revealed that SH003 target genes specifically regulate the necrosis process.

[0170]

[0171] Example 3: Evaluation of the mixed extract of Astragalus membranaceus, Angelica gigas, and Cnidium rhizome on the improvement of lung damage and alleviation of systemic inflammation using a mouse model.

[0172] 3-1. Experimental preparation and experimental method

[0173] All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyung Hee University (IACUC Approval No. KHSASP-22-131, Approval Date: 04 / 01 / 2022) and followed the ARRIVE guidelines. Twenty wild-type male C57BL / 6 mice (8 weeks old) were purchased from Daehan Biolink (Chungbuk, South Korea) and housed in a specific pathogen-free (SPF) animal center. The mice were randomly assigned to five groups. Two groups received oral administration of SH003 at 200 mg / kg or 400 mg / kg three times a week for 2 weeks. Then, 10 mg / kg of LPS from Escherichia coli O128:B12 (L2755, Sigma-Aldrich, St. Louis, MO, United States) in 50 μl of phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) to induce lung injury. Naïve mice were injected with 50 μl of PBS, and 5 mg / kg of dexamethasone (DEX) was administered intravenously 30 min before and after LPS injection as a positive control. After 24 h, the mice were euthanized by CO2 asphyxiation. After euthanasia, bronchoalveolar lavage (BAL) fluid, blood, spleen, and lung tissue were collected. The body weights of the mice were measured before LPS injection and at the time of sacrifice. For survival studies, 30 wild-type male C57BL / 6 mice (n = 10 per group) underwent the same procedure and were monitored for mortality for 7 days.

[0174] Mouse polymorphonuclear neutrophils (PMN) were isolated from mouse bone marrow or whole blood using Histopaque-1077 (Sigma-Aldrich) and Histopaque-1119 (Sigma-Aldrich). Isolated mouse PMN were pooled and the overall group average was presented.

[0175] Western blot analysis was performed throughout the experiment as follows. Lung tissues were lysed using RIPA buffer (Bioworld, Seoul, Korea). Tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The desired portion of the membrane was then excised and reacted with the appropriate primary and secondary antibodies. Endothelial nitric oxide synthase (eNOS) (#9572), NLRP3 (#8457), cleaved-gasdermin D (GSDMD) (#34,667), TDP-43 (TAR DNA-binding protein 43) (#3448), cleaved-caspase 1 (#89,332), absent in melanoma 2 (AIM2) (#63,660), cleaved-IL-1β (cleaved-1L-1β) (#63,124), cyclooxygenase-2 (COX-2) (#4842), nuclear factor kappa B (NF-κ) (#8242), p-NF-κB (Ser536) (#3033), extracellular signal-regulated kinase (ERK) (#4695), p-ERK (#4379), p38 (#8690), p-p38 (#4511), c-Jun N-terminal kinase (JNK) (#67,096), p-JNK (#4668), myeloperoxidase (MPO) (#79,623), arginase-1 (ARG1) (#93,668) antibodies were purchased from Cell Signaling Technology, Inc.(Beverly, MA, USA). Inducible nitric oxide synthase (iNOS) antibody (ab15323), peptidyl arginine deiminase 4 (PAD4) (ab96758), and citrullinated histone H3 (CitH3) (ab5103) were purchased from Abcam (Cambridge, UK). Neutrophil elastase (ELANE) antibody (AF4517) was purchased from R&D Systems (St. Louis, MO, USA), and GAPDH antibody (sc-32,233) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies, including anti-rabbit horseradish peroxidase (HRP) secondary antibody (HAF008), anti-mouse HRP secondary antibody (HAF007), and anti-rat HRP secondary antibody (HAF005), were purchased from R&D Systems, Inc. (St. Louis, MO, USA). Immunoblot signals were detected using Pierce™ECL Western Blotting Substrate (Waltham, MA, USA), and images were obtained using the X-ray film method or a Chemidoc imaging system (Davinch-K, Seoul, Republic of Korea). Relative protein levels were calculated using Image J software (Institutes of Health, Bethesda, MA, USA, https: / / imagej.nih.gov / ij / download.html, accessed on 27 April 2023) and visualized with GraphPad Prism 9.0 (GraphPad Software Inc., Boston, MA, USA).

[0176] H&E staining of lung sections was performed as described in a previous study (Antioxidants (Basel). 2021 Sep 5;10(9):1418). Representative images of lung sections were obtained by a blinded investigator. Immunohistochemical staining for CitH3 and immunofluorescence staining for NLRP3, ELANE, and CitH3 were performed as described previously, using antibodies against NLRP3 (A5652; Abclonal, Woburn, MA, USA), ELANE (A8953; Abclonal), or CitH3 (A18298; Abclonal). All images were visualized using an EVOS M7000 imaging system (Thermo Fisher Scientific).

[0177] Human and mouse polymorphonuclear neutrophils (PMNs) were isolated using a density gradient method. Specifically, mouse PMNs were isolated from mouse bone marrow or whole blood using Histopaque-1077 (Sigma-Aldrich) and Histopaque-1119 (Sigma-Aldrich). Isolated mouse PMNs were pooled and the overall group average was presented.

[0178]

[0179] 3-2. Lung damage relief assessment

[0180] Using an LPS-induced acute lung injury model, we studied whether SH003 affects acute lung injury mortality and morbidity.

[0181] First, the survival of mice with acute lung injury was evaluated. Mice prepared in Example 3-1 were administered 10 mg / kg of LPS to induce death. As shown in Figure 11, no mice in the naive group died by day 7. In contrast, no mice in the LPS-treated experimental group survived by day 7. Seven days after LPS injection, the mortality rate in the SH003 200 mg / kg group was 30%, and in the SH003 400 mg / kg group was 60%. The dexamethasone group, the positive control group, showed a survival rate of 40%. The mean survival times were 168 hours, 51.6 ± 11.3 hours, 90 ± 19.8 hours, 126 ± 19.9 hours, and 93.6 ± 21.3 hours in the no-treatment group, LPS-injected group, SH003 low-dose group (200 mg / kg), SH003 high-dose group (400 mg / kg), and positive control group (dexamethasone), respectively (Fig. 12). In addition, it was confirmed that SH003 administration could prevent weight loss induced by LPS injection in a dose-dependent manner (Fig. 13).

[0182] Lung damage was then assessed histologically. To determine the wet-to-dry ratio of mouse lung tissue, the wet weight of the lungs was measured immediately after excision. The lung tissues were then dried at 60°C for 24 hours, and the lung tissues were reweighed after drying to obtain a dry weight measurement. The wet-to-dry weight ratio was calculated by dividing the wet weight by the dry weight.

[0183] The wet / dry lung weight ratio, which indicates lung edema and damage, was significantly reduced by SH003 administration (Figs. 14 and 15). Mice administered SH003 showed reduced LPS-induced polymorphonuclear neutrophil (PMN) infiltration and damage (Figs. 14 and 16). Dexamethasone, the positive control, showed similar improvements to high-dose SH003.

[0184] Based on these results, we confirmed that administration of SH003 could improve lung damage and prevent death due to LPS-induced lung damage.

[0185]

[0186] 3-3. Evaluation of lung inflammation suppression

[0187] To evaluate the therapeutic efficacy of SH003, lung inflammation of the mice prepared in Example 3-1 was evaluated.

[0188] Whole mouse lungs were washed with 1.8 ml of PBS. To obtain BAL cells, the BAL solution was centrifuged at 500 × g for 5 min, and total inflammatory cells were counted using a hematology analyzer (Hemavet 950, Drew Scientific, Germany). BAL cells were mounted on microslides and stained with PROTOCOL Hema 3 fixative and solution (Fisher Scientific). The stained BAL cells were collected using a microscope (Zeiss Observer Z1, Carl Zeiss, Oberkochen, Germany).

[0189] Total BAL immune cell count (naive: 3.95 ± 1.29 × 10 5 / ml; LPS: 29.18 ± 5.64×10 5 / ml; LPS + SH003 200 mg / kg: 18.44 ± 2.40×10 5 / ml; LPS + SH003 400 mg / kg: 12.77 ± 1.97×10 5 / ml) was significantly decreased in SH003-treated mice depending on the concentration (Figs. 17 and 18). PMN count (Naive: 2.41 ± 0.43 x 10 5 / ml; LPS: 18.15 ± 3.80×10 5 / ml; LPS+SH003 200 mg / kg: 10.74 ± 1.11×10 5 / ml; LPS+SH003 400 mg / kg: 5.35 ± 1.47×105 / ml), macrophages (naive: 0.84 ± 0.42 × 10 5 / ml; LPS: 4.54 ± 1.97×10 5 / ml; LPS+SH003 200 mg / kg: 3.68 ± 1.08×10 5 / ml; LPS+SH003 400 mg / kg: 2.45 ± 0.60×10 5 / ml), lymphocytes (Naive: 0.88 ± 0.29 x 10 5 / ml; LPS: 3.94 ± 0.87×10 5 / ml; LPS+SH003 200 mg / kg: 2.88 ± 0.92×10 5 / ml; LPS+SH003 400 mg / kg: 2.42 ± 0.75×10 5 / ml) was significantly reduced in SH003-treated mice (Fig. 18).

[0190] In addition, SH003 was confirmed to have anti-inflammatory action. Specifically, cytokines and chemokines were measured using serum separated from whole blood of the mouse prepared in Example 3-1 and BAL solution collected. Cytokine and chemokine levels were measured using a custom multiplex panel configured to detect interleukin 1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-8, IL-18, monocyte chemoattractant protein-1 (MCP-1), chemokine (C-X-C motif) ligand 1 (CXCL1), and CXCL2. The analysis was performed using a Bio-Plex MAGPIX multiplex reader (Bio-Rad).

[0191] As a result, cytokines such as IL-1β, IL-6, and TNF-α (Fig. 19) and chemokines such as MCP-1, CXCL1, and CXCL2 (Fig. 20) were found to be reduced in the BAL solution.

[0192]

[0193] 3-4. Evaluation of systemic inflammation suppression

[0194] Systemic inflammation caused by sepsis is characterized by immunosuppression and a surge in cytokines and chemokines. This immune dysregulation phenomenon, commonly known as a cytokine storm, is closely associated with increased disease severity and a poor prognosis. Using the mice prepared in Example 3-1, cytokines and chemokines were measured and immune cell counts were determined using the same methods as in Examples 3-2 and 3-3.

[0195] In Example 3-1, it was confirmed that the plasma levels of cytokines, including IL-1β, IL-6, TNF-α, IFNγ, IL-8, and IL-18, significantly increased in mice with acute lung injury induced by LS administration. However, administration of SH003 suppressed the increase in these cytokine levels (Fig. 21). Furthermore, the increase in chemokines, including MCP-1, CXCL1, and CXCL2, was also suppressed by SH003 treatment (Fig. 22).

[0196] As a result, we confirmed that SH003 could improve the worsening of acute lung injury caused by sepsis by suppressing cytokine storm.

[0197] As with the number of immune cells in the BAL fluid, the increased number of PMNs in the circulation (Figure 23) confirmed that the inflammatory response was not only localized but also systemic. Persistent splenomegaly is another sign of a systemic response after sepsis induced by nonspecific infection or injury. As shown in Figure 24, the spleen was enlarged following LPS-induced sepsis.

[0198] Splenomegaly was inhibited by SH003 administration in a dose-dependent manner. PMNs have a short lifespan in the bloodstream, approximately 12–24 hours, and are supplied from the bone marrow (BM), particularly in inflammatory situations. Since a decrease in spleen size indirectly indicates a decrease in immune cell accumulation in the spleen, PMNs were analyzed in the BM of mice administered LPS.

[0199] As a result, as shown in Figure 25, low Ly6G expression was detected in BM-derived PMNs. Ly6G is a representative PMN-specific marker in mouse PMNs, and depending on its expression level, PMNs are mature Ly6 high PMN and immature Ly6G intermediate PMNs can be subdivided into circulating PMNs. In acute lung injury (ALI), PMNs rapidly migrate to the infected area, and the remaining PMNs in the BM are small and immature. High-dose SH003 treatment protected against LPS-induced changes in BM PMNs. LPS injection induced circulating PMNs to express CXCR4. hi -CXCR2 lo Although the surface marker movement was altered, mice administered SH003 showed a protective effect against this PMN characteristic change (Fig. 26).

[0200]

[0201] 3-5. Evaluation of inhibition of LPS-induced lung inflammation through the NF-κB and MAPK pathways

[0202] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are known to induce inflammation by producing mediators such as prostaglandins and nitric oxide (NO). As shown in Figure 27, SH003 suppressed the protein levels of iNOS and COX-2 induced by LPS. SH003 also suppressed the expression level of endothelial NOS (eNOS).

[0203] NF-κB activation plays a crucial role in the expression of iNOS, COX-2, and inflammatory cytokines. This transcription factor, NF-κB, regulates the activation of genes involved in the inflammatory and immune responses mediated by acute lung injury. We analyzed the levels of these proteins in lung tissue isolated from an acute lung injury model administered SH003.

[0204] As a result, administration of SH003 reduced LPS-induced NF-κB phosphorylation. Mitogen-activated protein kinase (MAPK) is also strongly involved in the expression of iNOS and COX-2. SH003 inhibited the phosphorylation of major MAPK family members, ERK, JNK, and p38.

[0205]

[0206] 3-6. Evaluation of inhibition of inflammasome activation in the lungs

[0207] Inflammasomes serve as an essential mechanism in the innate immune system to control various inflammatory diseases by releasing IL-1β. To elucidate the signaling pathways that regulate inflammasome activation, Western blotting was performed using lung tissue from the mouse model prepared in Example 3-1.

[0208] First, we examined the expression level of the NLRP3 inflammasome, the most characteristic sensor protein in the inflammasome family. NLRP3 is known to be activated by various stimuli, including pathogens, environmental toxins, and endogenous danger signals. As shown in lung tissue staining, the LPS-induced increase in NLRP3 in the lungs was suppressed by SH003 administration (Figure 28). Furthermore, SH003 administration reduced NLRP3 and related protein levels, as well as cleaved GSDMD, TDP-43, cleaved caspase-1, and cleaved IL-1β. SH003 also suppressed inflammasome AIM2 and NLRP3 protein levels (Figure 29).

[0209] These results indicate that SH003 can suppress the inflammatory response in acute lung injury by regulating the inflammasome signaling cascade.

[0210]

[0211] 3-7. Evaluation of inhibition of NET formation in the lungs

[0212] Neutrophil extracellular traps (NETs) are formed by various stimuli, including pathogens, cytokines, and microcrystals. NETs are composed of DNA, histones (citrullinated), chromatin, and several enzyme proteins, such as MPO or neutrophil elastase (ELANE).

[0213] SH003 was used to confirm whether it could regulate NETosis-related markers, including PAD4, MPO, ELANE, ARG-1, and CitH3, in the lungs of acute lung injury mice using the mouse model of Example 3-1.

[0214] As a result, SH003 was confirmed to significantly reduce the protein expression of NETosis-related markers (Fig. 30). Furthermore, the NET inhibitory effect of SH003 was confirmed through CitH3 staining in lung tissue. Furthermore, the LPS-induced increase in ELANE was suppressed by SH003 treatment (Fig. 31).

[0215]

[0216] Example 4: Evaluation of the improvement of lung damage by a mixed extract of Astragali, Angelica, and Cnidium officinalis using human polymorphonuclear neutrophils (hPMNs)

[0217] 4-1. NET formation inhibition evaluation

[0218] To determine whether SH003 could be applied to humans, PMNs isolated from human blood were used.

[0219] For human PMNs (hPMNs), the research protocol involving the use of human blood cells was approved by the Institutional Review Board of Kyung Hee University (IRB approval number: KHSIRB-22-535, approval date: November 7, 2022). hPMNs were isolated from healthy donors using PolymorphPrep (1895; Alere Technologies AS, Oslo, Norway) with written consent.

[0220] Reactive oxygen species (ROS) were measured using luminol-dependent chemiluminescence in a luminometer (Glomax Discover, Promega). hPMNs were pretreated with 40 μg / ml mtDNA or the same volume of Tris-EDTA buffer (vehicle) for 15 min with rotation at RT. Cells (4 × 10 6 (cells / ml) were mixed 1:1 with 2× detection reagent consisting of 0.2 mM luminol (Sigma, 123,072) and 150 nM HRP in DPBS+ (14,040,117; Gibco) and preheated at 37°C for 5 min. After preheating, 180 μl of the hPMN / detection reagent mixture was manually transferred in quadruplicate to a preheated 96-well plate, and chemiluminescence detection was started. At the indicated time points, 20 μl of DPBS+ (resting cells) or 5 μM phorbol 12-myristate 13-acetate (PMA) was automatically injected by the luminometer. SH003 were treated in maintenance medium for 60 min before starting the assay. Output is expressed in relative luminous units per second (RLU / sec) and quantified as the area under the curve (AUC) over the displayed period.

[0221] To stain NETs, ​​freshly isolated hPMNs were cultured with SH003 for 60 min and then stimulated with 1 μg / ml LPS in RPMI 1640 medium in a rotating tube at 37°C for 1 h. The hPMNs were then plated on poly-l-lysine-coated 4-well slides. NET formation was confirmed by cell staining for CitH3 (A18298; Abclonal). After washing, Alexa 488-conjugated (A11070; Thermo Fisher Scientific) secondary antibody was applied, and cells were counterstained with DAPI (Sigma-Aldrich). Images were acquired with an EVOS M7000 imaging system (Thermo Fisher Scientific). Quantitative NET analysis was performed using the elastase technique (601010; Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. hPMNs were cultured in NET assay buffer containing SH003 for 60 min, then treated with 20 nM PMA for 4 h at 37°C to induce forced NET formation. The supernatant was transferred to a microtube, nuclease inactivated with ethylenediaminetetraacetic acid, and centrifuged to remove debris. Subsequently, elastase levels (mU / ml) were measured by adding substrate (N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide) to the samples, using a standard curve generated using the provided neutrophil elastase reagent.

[0222] Control groups were cultured in PBS for the same time, and SH003 were pretreated with doses of 100 and 200 μg / ml 30 min before PMA exposure.

[0223] As a result, SH003 treatment was shown to prevent excessive NET formation by suppressing PMA-induced CitH3 expression (Fig. 32). Similar to the in vivo results, LPS induced an increase in the NET-associated proteins MPO and ELANE, along with the inflammasome marker NLRP3, which was reduced by SH003 treatment (Fig. 33).

[0224] Additionally, elastase levels were measured in the maintenance medium of hPMN. As a result, high doses of SH003 were found to significantly reduce elastase levels (Fig. 34).

[0225] ROS, along with DNA and various enzymes, are components of NETs. SH003 treatment inhibited ROS production in response to PMA, indicating inhibition of NET formation by SH003 (Fig. 35).

[0226] Consequently, we confirmed that SH003 is effective against hPMNs exposed to bacterial endotoxin LPS or protein kinase C agonist PMA by modulating multiple pathways, inducing immune response pathways including NETosis.

[0227]

[0228] 4-2. NET-related path suppression evaluation

[0229] To determine which pathways are involved in the protective effect of SH003 on acute lung injury, mass cytometry by time-of-flight (CyTOF) was performed on LPS-treated hPMNs.

[0230] For mass cytometry, hPMNs were isolated and prepared at 1 × 107 cells / ml in MaxPar PBS (Fluidigm, South San Francisco, CA, USA). hPMNs were exposed to LPS at a concentration of 1 μg / ml for 30 min. Prior to LPS exposure, hPMNs were pretreated with SH003 for 60 min. Viability was then determined by adding Cell-ID cisplatin working solution (Fluidigm) for 5 min. Cells were washed and resuspended in MaxPar cell staining buffer (Fluidigm). In subsequent immunostaining steps, antibodies for surface markers conjugated to metal isotopes (using the MaxPar reagent kit from Fluidigm) were used on the cells for 30 min. Cells were then fixed and permeabilized using MaxPar Fix I buffer (Fluidigm) and 1× Barcode Permanent buffer (Fluidigm), respectively. Antibodies for intracellular markers conjugated with metal isotopes were added to the cells for 30 minutes. Stained hPMNs were washed with MaxPar cell staining buffer, fixed with methanol and 1.6% formaldehyde solution, centrifuged, and the supernatant was removed. Cell-ID™ intercalator (Fluidigm) was added overnight at 4°C. The following day, cells were centrifuged and washed with MaxPar cell collection solution (Fluidigm). CyTOF analysis was then performed using a Helios CyTOF mass cytometer (Fluidigm).

[0231] As a result, we confirmed that LPS had a significant effect on the expression of PMN markers through the FlowSOM tree (Fig. 36) and t-distributed Stochastic Neighbor Embedding (tSNE) technique (Fig. 37). SH003 pretreatment showed a protective effect against changes in the corresponding markers induced by LPS. The heatmap displays detailed results for each marker (Fig. 38). NET-related factors such as ARG1 and C5a receptor CD88 were restored to normal by SH003, and the increase in NLRP3 induced by LPS was also suppressed (Fig. 39).

[0232] These results indicate that the protective effect of SH003 on septic acute lung injury is closely related to the inflammasome pathway, and that the pathway related to NET formation is increased by LPS exposure, but SH003 reverses this change.

[0233]

[0234] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical concept or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modified forms derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A pharmaceutical composition for preventing or treating respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

2. In paragraph 1, The above extract is a pharmaceutical composition in which Astragalus membranaceus, Angelica gigas, and Cnidium rhizome are each extracted with at least one solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

3. In paragraph 2, A pharmaceutical composition, wherein the alcohol having 1 to 4 carbon atoms is 20 to 40% (v / v) ethanol.

4. In any one of paragraphs 1 to 3, A pharmaceutical composition wherein the above mixed extracts of Astragali, Angelica gigas, and Cnidium rhizome are mixed in a weight ratio of Astragali: Angelica gigas: Cnidium rhizome = 0.5 to 5: 0.5 to 1.5: 0.5 to 1.

5.

5. In any one of paragraphs 1 to 4, A pharmaceutical composition, wherein the above respiratory inflammatory disease is at least one selected from the group consisting of acute lung injury, emphysema, asthma, chronic obstructive pulmonary disease, otitis media, sinusitis, tonsillitis, nasopharyngitis, laryngitis, and acute bronchitis.

6. In any one of paragraphs 1 to 5, A pharmaceutical composition wherein the above respiratory inflammatory disease is caused by sepsis.

7. In any one of paragraphs 1 to 6, A pharmaceutical composition wherein the composition suppresses systemic inflammation.

8. In any one of paragraphs 1 to 7, A pharmaceutical composition, wherein the composition suppresses lung inflammation.

9. In any one of paragraphs 1 to 8, A pharmaceutical composition wherein the composition inhibits NF-κB and mitogen-activated protein kinase phosphorylation.

10. In any one of paragraphs 1 to 9, A pharmaceutical composition, wherein the composition inhibits inflammasome activation and neutrophil extracellular trap formation.

11. A method for preventing or treating respiratory inflammatory disease or sepsis, comprising administering to a subject a composition of any one of claims 1 to 10.

12. A food composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

13. A pharmaceutical composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

14. A feed composition for preventing or improving respiratory inflammatory disease or sepsis, comprising a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowiiMaximowicz as an active ingredient.

15. Use of a composition comprising a mixed extract of Astragali, Angelica and Cnidium officinalis for the prevention or treatment of respiratory inflammatory diseases or sepsis.

16. Use of a composition comprising a mixed extract of Astragali, Angelica and Cnidium officinalis for the manufacture of a medicament for the prevention or treatment of respiratory inflammatory diseases or sepsis.

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