Composition for preventing or treating neuroinflammation comprising extract of aerial parts of peucedanum japonicum as active ingredient

Abstract

The present invention relates to a composition for preventing or treating neuroinflammation, comprising, as an active ingredient, an extract of the aerial parts of Peucedanum japonicum. The extract of the aerial parts of Peucedanum japonicum and a compound isolated therefrom, of the present invention, were confirmed to inhibit NO production in microglial cell lines in which inflammatory responses are induced by LPS. It was also confirmed that the extract of the aerial parts of Peucedanum japonicum inhibited various inflammatory pathways related to neuroinflammation, and inhibited inflammatory activation factors. Furthermore, the extract was confirmed to inhibit the expression of inflammatory proteins in neuroinflammatory responses, thereby confirming the anti-neuroinflammatory effect thereof.

Description

A composition for the prevention or treatment of brain inflammation containing an extract of the above-ground parts of Saposhnikovia divaricata as an active ingredient

[0001] The present application claims priority based on Korean Patent Application No. 10-2024-0165541 filed on November 19, 2024, and all contents disclosed in the specification and drawings of said application are incorporated by reference into the present application.

[0002] The present invention relates to a composition for the prevention or treatment of encephalitis comprising an extract of the above-ground parts of Saposhnikovia divaricata as an active ingredient.

[0003] Microglia are a type of immune cell that function as brain macrophages, playing a crucial role in host defense and tissue repair within the central nervous system (CNS). Under normal conditions, glial cells (i.e., astrocytes and microglia) support and protect neurons from pathogens and maintain synaptic homeostasis. Microglia are primarily found in the hippocampus and cortex, which govern learning and memory. Abnormal activation of microglia by external stimuli leads to a wide range of responses, including the initiation of inflammation, the secretion of pro-inflammatory mediators, and the release of neurotoxic factors and various cytokines; this process is involved in all neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and stroke. Therefore, inhibiting the abnormal activation of microglia may hold valuable therapeutic potential for the treatment of inflammation-related diseases. BV2 microglia have been widely used as a cellular model of neuroinflammation to investigate inflammation-promoting cytokines and responses to oxidative stress for the search for new drug candidates.

[0004] Lopolysacharide (LPS) is a well-known potent microglia activator that acts as a typical endotoxin causing inflammation, septic shock, and death. Therefore, LPS is commonly used as an in vitro model of inflammation. LPS generally induces inflammation through two signaling pathways: the NF (nuclear factor)-kB and MAPK (mitogen-activated protein kinase) pathways. The MAPK family, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH2-terminal kinase), and p38, is involved in regulating the secretion of inflammatory factors within activated microglia. This signaling pathway sequentially mediates transcription factors such as NF-kB. The activating transcription factor NF-kB is translocated to the nucleus and stimulates the expression of several neurotoxic factors in glial cells, including iNOS, COX-2, and pro-inflammatory cytokines. LPS-induced activation of microglia triggers the release of TNF-α (tumor necrosis factor α) and promotes TNF-α or NO-mediated apoptosis. Therefore, MAPK and NF-kB pathways are considered potential therapeutic targets for neuroinflammatory diseases. Many therapeutic agents exert anti-neuroinflammatory effects by inhibiting MAPK and NF-kB.

[0005] Meanwhile, *Peucedanum japonicum*, also known as sea parsley, is a plant of the Apiaceae family that grows naturally in the temperate climates of the south, including Jeju Island and Ulleungdo. The young shoots, tender leaves, fruits, and roots of *Peucedanum japonicum* are all edible, and the roots are used medicinally under the names *Bangpung*, *Bangpungyeop*, and *Bangpunghwa*. The medicinal effects of *Bangpung* include diaphoretic, anti-wind, dampness-removing, and pain-relieving properties. *Bangpungyeop* is used to treat sweating caused by heat due to stroke, while *Bangpunghwa* is used to treat abdominal pain, acute pain in the limbs, difficulty moving, pain in the lower back and meridians, and pain between muscles and bones.

[0006] Accordingly, the inventors confirmed that the extract of the above-ground parts of Saposhnikovia divaricata has an effect on improving brain inflammation, and thus completed the present invention.

[0007] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of encephal inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0008] Another objective of the present invention is to provide a food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0009] Another objective of the present invention is to provide a health functional food composition for the prevention or improvement of brain inflammatory diseases, comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0010] Another objective of the present invention is the step of drying the above-ground parts of the *Saposhia divaricata*;

[0011] A step of extracting the above-mentioned dried above-ground parts of *Saposhia divaricata* with a solvent;

[0012] The present invention provides a method for manufacturing a food composition for the prevention or improvement of brain inflammatory diseases.

[0013] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of encephal inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0014] In addition, the present invention provides a food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0015] In addition, the present invention provides a health functional food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0016] In addition, the present invention comprises the step of drying the above-ground parts of the *Sophora japonica* plant;

[0017] A step of extracting the above-mentioned dried above-ground parts of *Saposhia divaricata* with a solvent;

[0018] A method for manufacturing a food composition for the prevention or improvement of brain inflammatory diseases is provided.

[0019] It was confirmed that the above-ground extract of *Saposhia divaricata* and compounds isolated therefrom of the present invention inhibit NO production in microglia cell lines in which an inflammatory response was induced by LPS. Furthermore, it was confirmed that the above-ground extract of *Saposhia divaricata* inhibits various inflammatory pathways associated with brain inflammation and inhibits inflammatory activating factors. Additionally, by confirming that it inhibits the expression of inflammatory proteins in brain inflammatory responses, an anti-brain inflammatory effect was verified, which can be usefully applied in related industries.

[0020] Figure 1 is a figure showing the structure of a compound isolated from the above-ground extract of Saposhnikovia divaricata according to the present invention.

[0021] Figure 2 is a figure confirming the NO production inhibitory effect of a compound isolated from the above-ground part extract of Saposhnikovia divaricata of the present invention.

[0022] Figure 3 is a figure confirming the cell viability of the above-ground extract of Saposhnikovia divaricata according to the present invention (A: confirmation of water extract, B: confirmation of ethanol extract).

[0023] Figure 4 is a figure confirming the NO production inhibitory effect of the above-ground extract of Saposhnikovia divaricata according to the present invention (A: confirmation of water extract, B: confirmation of ethanol extract).

[0024] Figure 5 is a Western blot analysis of the NF-κB signaling pathway inhibitory effect of the ethanol extract of the above-ground parts of Saposhnikovia divaricata according to the present invention (A: confirmation of IκB phosphorylation, B: confirmation of p65 and p50 nuclear translocations).

[0025] Figure 6 is a Western blot analysis of the inhibitory effect of the ethanol extract of the above-ground parts of Saposhnikovia divaricata according to the present invention on the MAPK signaling pathway (A: confirmation of p38 phosphorylation, B: confirmation of ERK phosphorylation, C: confirmation of JNK phosphorylation).

[0026] Figure 7 is a Western blot analysis of the inhibitory effects of the ethanol extract of the above-ground parts of Saposhnikovia divaricata according to the present invention on the Akt and GSK3β signaling pathways (A: confirmation of Akt phosphorylation, B: confirmation of GSK3β phosphorylation).

[0027] Figure 8 is a Western blot analysis of the inhibitory effect of the ethanol extract of the above-ground parts of Saposhnikovia divaricata according to the present invention on the JAK2 / STAT3 signaling pathway (A: confirmation of JAK2 phosphorylation, B: confirmation of STAT3 phosphorylation).

[0028] Figure 9 is a figure confirming the inhibitory effect of the ethanol extract of the above-ground parts of Saposhnikovia divaricata of the present invention on the expression of inflammatory activating factors (A: Quantification of TLR4 expression, B: Quantification of MyD88 expression).

[0029] Figure 10 is a figure confirming the expression of inflammatory proteins in the ethanol extract of the above-ground parts of Saposhnikovia divaricata according to the present invention (A: quantification of PGE2 production, B: confirmation of iNOS and COX-2 expression).

[0030] Figure 11 is a figure confirming the expression of inflammatory cytokines of the ethanol extract of the above-ground parts of Saposhnikovia divaricata according to the present invention.

[0031] A: Quantification of IL-6 production

[0032] B: Quantification of TNF-α production

[0033] C: Quantification of IL-6 mRNA expression

[0034] D: Quantification of TNF-α mRNA expression

[0035] Embodiments of the present invention will be described in detail below with reference to the attached drawings. In the following description, detailed descriptions of technologies well known to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such descriptions would unnecessarily obscure the essence of the present invention. Additionally, the terminology used in this specification is used to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs.

[0036] Therefore, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0037] The present invention provides a pharmaceutical composition for the prevention or treatment of encephal inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0038] The term “prevention” as used in this invention refers to any act of suppressing the symptoms of a specific disease or delaying its progression through the administration of the composition of this invention.

[0039] The term “treatment” as used in this invention refers to any act of improving or beneficially altering the symptoms of a specific disease through the administration of the composition of this invention.

[0040] The pharmaceutical composition of the present invention may additionally include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete or incomplete adjuvant may be further included to increase the effect.

[0041] The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited to these, but may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0042] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, or sterile injectable solutions, each according to conventional methods.

[0043] When formulating, the product may be prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and such solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the active ingredient. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Water-insoluble solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Bases for suppositories may include Witepsol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc.

[0044] The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration may be anticipated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0045] The dosage of the pharmaceutical composition according to the present invention is selected by taking into consideration the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and preferably, it is included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg / ml, it may exhibit toxicity to the human body.

[0046] The "above-ground part" of the present invention refers to the part of a plant body located above the surface of the earth, and includes all parts such as the plant stem and leaves, flowers, and fruits attached to the stem, and is referred to as a shoot system.

[0047] According to one embodiment of the present invention, the extract may be extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohols, aqueous solutions of lower alcohols, nucleic acids, chloroform, ethyl acetate, and acetone, preferably water or ethanol, and more preferably ethanol, but is not limited thereto.

[0048] According to one embodiment of the present invention, the extract may comprise a compound selected from the group consisting of compounds represented by the following chemical formulas 1 to 5.

[0049] [Chemical Formula 1]

[0050]

[0051] [Chemical Formula 2]

[0052]

[0053] [Chemical Formula 3]

[0054]

[0055] [Chemical Formula 4]

[0056]

[0057] [Chemical Formula 5]

[0058]

[0059] According to one embodiment of the present invention, the extract may inhibit NO production.

[0060] According to one embodiment of the present invention, the extract may inhibit phosphorylation or activity of an inflammatory pathway, and the inflammatory pathway may be a pathway selected from the group consisting of NF-κB (Nuclear Factor Kappa B), MAPK (Mitogen-activated protein kinase), Akt (Protein kinase B), GSK3β (Glycogen synthase kinase-3 beta), JAK2 (Janus Kinase 2), and STAT3 (Signal transducer and activator of transcription 3).

[0061] According to one embodiment of the present invention, the NF-κB pathway may include IκB (IkappaB kinase), and

[0062] According to one embodiment of the present invention, it may include an intranuclear potential of p65 or p50.

[0063] According to one embodiment of the present invention, the MAPK pathway may include p38, ERK (extracellular regulated protein kinase) or JNK (c-jun N-terminal kinase).

[0064] According to one embodiment of the present invention, the extract may inhibit the expression of an inflammatory pathway activating factor, and the inflammatory pathway activating factor may be TLR4 (Toll-like receptor 4) or MYD88 (Myeloid differentiation primary response 88).

[0065] According to one embodiment of the present invention, the extract may inhibit the expression of an inflammatory protein, and the inflammatory protein may be PGE2 (prostaglandin E2), iNOS (Inducible Nitric Oxide Synthase), or COX-2 (Cyclooxygenase-2).

[0066] According to one embodiment of the present invention, the extract may inhibit the expression of inflammatory cytokines, and the inflammatory cytokines may be IL-6 or TNF-α (tumor necrosis factor-α).

[0067] According to one embodiment of the present invention, the brain inflammatory disease may be a disease selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, neurological autoimmune disease, inflammatory and neuropathic pain, and cerebrovascular disease.

[0068]

[0069] In addition, the present invention provides a food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0070] As used in the present invention, the term “improvement” refers to any action that at least reduces parameters related to the condition being treated, such as the degree of symptoms.

[0071] In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, as in conventional food compositions.

[0072] Examples of the natural carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The flavoring agents described above may advantageously use natural flavoring agents (taumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition described above and used as a functional food or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0073] In addition, the above food composition may contain, in addition to the extract which is an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

[0074] The functional food composition of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., for the purpose of preventing or treating brain inflammatory diseases. In the present invention, the term "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body pursuant to Article 6727 of the Act on Health Functional Foods, and means consuming it for the purpose of obtaining effects useful for health uses, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention may include conventional food additives, and unless otherwise stipulated, suitability as a food additive is determined according to the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Items listed in the above "Food Additives Codex" include, for example, chemical synthetic compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline additives for noodles, preservative preparations, and tar dye preparations. For example, a health functional food in tablet form may be produced by granulating a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives using a conventional method, and then adding a lubricant or the like and compression molding, or by directly compression molding the said mixture. In addition, the health functional food in tablet form may contain a binder or the like as needed. Among health functional foods in capsule form, hard capsules may be manufactured by filling a conventional hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules may be manufactured by filling a capsule base such as gelatin with a mixture of the active ingredient of the present invention mixed with additives such as excipients. The above soft capsule may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.A health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., using a previously known method, and if necessary, it can be coated with sucrose or other coating agents, or the surface can be coated with a substance such as starch or talc. A health functional food in the form of a granule can be prepared by making a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., into a granular form using a previously known method, and may contain flavoring agents, stimulating agents, etc., if necessary.

[0075]

[0076] In addition, the present invention provides a health functional food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

[0077]

[0078] In addition, the present invention comprises the step of drying the above-ground parts of the *Sophora japonica* plant;

[0079] A step of extracting the above-mentioned dried above-ground parts of *Saposhia divaricata* with a solvent;

[0080] A method for manufacturing a food composition for the prevention or improvement of brain inflammatory diseases is provided.

[0081] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0082] <Preparation Example 1> Preparation of extract from the above-ground parts of *Saposhnikovia divaricata*

[0083] An extract of the above-ground parts of Saposhnikovia divaricata according to the present invention was prepared. Specifically, 10 cm of the apical (terminal) part of the stem of the above-ground parts of Saposhnikovia divaricata was obtained and dried. Then, the dried above-ground parts of Saposhnikovia divaricata were used to prepare an extract by repeating the process a total of two times at a temperature of 60°C to 80°C using hot water (PJW), 30% ethanol (PJE), or 50% ethanol, with a solvent ratio of 8 to 10 times the volume of the sample. The yield of the extract obtained at this time was confirmed to be approximately 29% to 35%.

[0084]

[0085] <Preparation Example 2> Isolation of compounds derived from extract of the above-ground parts of Saposhnikovia divaricata

[0086] The present invention aimed to isolate compounds derived from the above-ground extract of *Saposhnikovia divaricata*. From 30 g of the 30% ethanol extract obtained in Preparation Example 1, ethyl acetate, n-butanol, and water were used to obtain approximately 10 g of ethyl acetate, 7 g of the n-butanol fraction, and 13 g of the water fraction. Subsequently, the obtained ethyl acetate fraction was purified using SiO2, ODS, and MPLC equipment, and five compounds with a sesquiterpene lactone structure were isolated.

[0087] [Chemical Formula 1]

[0088]

[0089] [Chemical Formula 2]

[0090]

[0091] [Chemical Formula 3]

[0092]

[0093] [Chemical Formula 4]

[0094]

[0095] [Chemical Formula 5]

[0096]

[0097]

[0098] <Example 1> Confirmation of NO production inhibition by a compound derived from Saposhnikovia divaricata

[0099] We confirmed whether the compound derived from the above-ground parts of *Saposhia divaricata* according to the present invention inhibits NO production in microglia cell lines in which a brain inflammatory response was induced. Specifically, the amount of NO secreted into the medium from BV2 cell lines was measured using Griess reagent, and BV2 cells were placed in a 24-well plate at a volume of 2×10 5 After inoculating at a concentration of cell / mL, the five compounds isolated in Preparation Example 2 were inoculated at concentrations of 10, 20, 40, and 80 μM, respectively, and reacted for 3 hours. Subsequently, LPS (1 μg / mL) was added, and the mixture was cultured for 24 hours. After the culture was completed, 100 μL of culture medium was reacted with an equal volume of Griess reagent (SigmaAlderich, St. Louis, MO) for 15 minutes, and the absorbance was measured at 540 nm using a microplate reader. The concentration of NO was measured based on the sodium nitrite standard curve. As controls, an untreated control group (Control) and an LPS group treated with LPS alone were used.

[0100]

[0101] As a result, as shown in Figure 2, compared to the control group, NO production was significantly increased in the group treated with LPS, but it was confirmed that all five compounds derived from Saposhnikovia divaricata inhibited NO production in a concentration-dependent manner.

[0102]

[0103] <Example 2> Confirmation of Cytotoxicity of Extract from Above-Ground Parts of Saposhnikovia divaricata

[0104] The cytotoxicity of the above-ground extract of Saposhnikovia divaricata according to the present invention was confirmed. Specifically, 1.0 × 10⁻⁶ in a 96-well plate 5BV2 cell lines were cultured at a concentration of cells / mL, then treated with PJE at concentrations of 12.5, 25, 50, 100, and 200 μg / mL, respectively, and cultured for 24 hours. Afterward, MTS [3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Promega, Madison, WI] was treated for 1 hour, and the absorbance was measured at 490 nm using a multi-plate reader (Biotek, VT, USA).

[0105]

[0106] As a result, as shown in Figure 3, it was confirmed that the ethanol extract of the above-ground parts of the *Saposhia divaricata* of the present invention had no cytotoxicity up to a concentration of 100 μg / ml, and subsequent experiments were conducted at a concentration of 100 μg / ml.

[0107]

[0108] <Example 3> Confirmation of NO production inhibition by above-ground extract of Saposhnikovia divaricata

[0109] It was confirmed whether the above-ground extract of Saposhnikovia divaricata of the present invention inhibits NO production in LPS-induced brain inflammation. Specifically, NO production was confirmed in BV2 cell lines using the same method as in Example 1 above, and was confirmed by treating with PJE at concentrations of 25, 50, and 100 μg / ml.

[0110]

[0111] As a result, as shown in Figure 4, LPS-induced NO production was significantly reduced by treatment with PJE, confirming that extracting the above-ground parts of Saposhnikovia divaricata with ethanol as a solvent has an excellent effect in inhibiting brain inflammatory responses.

[0112]

[0113] <Example 4> Confirmation of Inhibition of Brain Inflammation Pathway by Extract of Above-Ground Parts of Saposhnikovia divaricata

[0114] It was confirmed whether the above-ground extract of *Saposhia divaricata* of the present invention inhibits brain inflammation pathways. Specifically, proteins were extracted from each group of BV2 cell lines cultured in Example 3 by adding RIPA buffer (ThermoScientific, MA). To measure the inhibitory activity of phosphorylation of IκB (IkappaB kinase) protein in the NF-κB (Nuclear Factor Kappa B) pathway and the inhibitory activity of nuclear translocation of p65 and p50, proteins were extracted from BV2 cells by adding RIPA buffer (ThermoScientific, MA), and protein quantification was performed using the Bradford protein assay (Bio-Rad Laboratories). Equal amounts of protein were then electrophoresed on 7.5% and 12% SDS-polyacrylamide gels and transcribed onto a nitrocellulose membrane (NC membrane). After blocking the transcribed NC membrane in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline) containing 5% non-fat oil, p-IκBα, β-actin, nuclear p65, and PCNA antibodies were added at a 1:1000 ratio and incubated for 12 hours at 4°C. Subsequently, secondary antibodies (anti-mouse, anti-rabbit IgG) were added at a 1:1000 ratio and incubated for 1 hour. Finally, an ECL solution mixed thoroughly at a 1:1 ratio was poured onto the NC membrane to induce luminescence, and protein expression was confirmed using a ChemiDocMP System (Bio-Rad). Additionally, nuclear translocation reactions were confirmed by the phosphorylation of p65 and p50.

[0115] In addition, the phosphorylation of p38, ERK (extracellular regulated protein kinase), and JNK (c-jun N-terminal kinase) of the MAPK (Mitogen-activated protein kinase) pathway, the phosphorylation of Akt (Protein kinase B) and GSK3β (Glycogen synthase kinase-3 beta) pathways, and the phosphorylation of JAK2 (Janus Kinase 2) and STAT3 (Signal transducer and activator of transcription 3) of the brain inflammatory response were confirmed using the same method as above.

[0116]

[0117] As a result, as shown in Figure 5, it was confirmed that PJE inhibits LPS-induced phosphorylation of IκB in a concentration-dependent manner. In addition, it was confirmed that the nuclear p65 and p50 potentials increased by LPS decrease in a concentration-dependent manner.

[0118]

[0119] In addition, phosphorylation of p38, ERK, and JNK of the MAPK pathway was induced by LPS, but it was confirmed that phosphorylation of proteins of the MAPK pathway was inhibited when PJE was treated (Fig. 6).

[0120]

[0121] In addition, treatment with LPS increased the phosphorylation of the Akt and GSK3β pathways and the phosphorylation of JAK2 and STAT3, but it was confirmed that the PJE of the present invention inhibited the phosphorylation of Akt, GSK3, JAK2, and STAT3 in a concentration-dependent manner (Figs. 7 and 8), and it was confirmed that the ethanol extract of the above-ground parts of Saposhnikovia divaricata of the present invention improves the brain inflammatory response by inhibiting various inflammatory pathways in the brain inflammatory response.

[0122]

[0123] <Example 5> Confirmation of Inhibition of Inflammatory Activating Factors by Extract of Above-Ground Parts of Saposhnikovia divaricata

[0124] It was confirmed whether the above-ground extract of *Saposhia divaricata* of the present invention inhibits the expression of TLR4 (Toll-like receptor 4), an inflammatory response activation factor of immune cells, and MyD88 (Myeloid differentiation primary response 88), a TLR4 stimulator. Specifically, RNA was extracted from the cell lines of each group of Example 3 using TRI reagent (Molecular Research Center Inc.), and mRNA expression was confirmed using qRT-PCR on the cDNA obtained by reverse transcribing the RNA.

[0125]

[0126] As a result, as shown in Figure 9, the expression of TLR4 and MyD88 was significantly increased in BV2 cell lines stimulated with LPS, but it was confirmed that the expression of TLR4 and MyD88 decreased in a concentration-dependent manner in the group treated with the PJE of the present invention.

[0127]

[0128] <Example 6> Confirmation of Inhibition of Inflammatory Proteins by Above-Ground Extract of Saposhnikovia divaricata

[0129] It was confirmed whether the above-ground extract of *Saposhia divaricata* of the present invention inhibits the expression of inflammatory proteins PGE2 (prostaglandin E2), iNOS (Inducible Nitric Oxide Synthase), and COX-2 (Cyclooxygenase-2). Specifically, the expression of PGE2 was quantified by obtaining a cell culture medium and analyzing it with ELISA, and the protein expression of iNOS and COX-2 was analyzed by Western blotting in the same manner as in Example 4.

[0130]

[0131] As a result, as shown in Figure 10, the expression of PGE2, iNOS, and COX-2 was significantly increased by treatment with LPS, but when PJE was treated, the increased expression of PGE2, iNOS, and COX-2 was reduced in a concentration-dependent manner.

[0132]

[0133] <Example 7> Confirmation of Inhibition of Inflammatory Cytokine Expression by Extract of Above-Ground Parts of Saposhnikovia divaricata

[0134] It was confirmed whether the above-ground extract of *Saposhnikovia divaricata* of the present invention inhibits the production of inflammatory cytokines IL-6 and TNF-α (tumor necrosis factor-α) and mRNA expression. Specifically, the production of IL-6 and TNF-α was confirmed using an ELISA kit with the cell culture medium of Example 3, and mRNA expression was confirmed in the same manner as in Example 5.

[0135]

[0136] As a result, as shown in Figure 11, the production of inflammatory cytokines IL-6 and TNF-α increased in cells treated with LPS, but it was confirmed that the increased production of IL-6 and TNF-α was significantly reduced when treated with PJE. In addition, it was confirmed that PJE inhibited the mRNA expression of IL-6 and TNF-α.

[0137]

[0138] Accordingly, it was confirmed that the above-ground extract of *Saposhnikovia divaricata* and compounds isolated therefrom of the present invention inhibit NO production in microglia cell lines in which an inflammatory response was induced by LPS. Furthermore, it was confirmed that the above-ground extract of *Saposhnikovia divaricata* inhibits various inflammatory pathways associated with brain inflammation and inhibits inflammatory activating factors. Additionally, by confirming that it inhibits the expression of inflammatory proteins in brain inflammatory responses, an anti-brain inflammatory effect was verified.

Claims

1. A pharmaceutical composition for the prevention or treatment of encephal inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

2. In Paragraph 1, A composition wherein the above extract is extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohols, aqueous solutions of lower alcohols, hexane, chloroform, ethyl acetate, and acetone.

3. In Paragraph 1, A composition wherein the extract comprises a compound selected from the group consisting of compounds represented by the following chemical formulas 1 to 5. [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] 4. In Paragraph 1, The above extract is a composition that inhibits NO production.

5. In Paragraph 1, A composition in which the above extract inhibits phosphorylation or activity of an inflammatory pathway.

6. In Paragraph 5, A composition wherein the above inflammatory pathway is a pathway selected from the group consisting of NF-κB (Nuclear Factor Kappa B), MAPK (Mitogen-activated protein kinase), Akt (Protein kinase B), GSK3β (Glycogen synthase kinase-3 beta), JAK2 (Janus Kinase 2), and STAT3 (Signal transducer and activator of transcription 3).

7. In Paragraph 6, A composition in which the above NF-κB pathway includes IκB (IkappaB kinase).

8. In Paragraph 6, A composition in which the above NF-κB pathway includes an intranuclear potential of p65 or p50.

9. In Paragraph 6, A composition wherein the above MAPK pathway comprises p38, ERK (extracellular regulated protein kinase) or JNK (c-jun N-terminal kinase).

10. In Paragraph 1, A composition in which the above extract inhibits the expression of inflammatory pathway activating factors.

11. In Paragraph 10, A composition in which the inflammatory pathway activating factor is TLR4 (Toll-like receptor 4) or MYD88 (Myeloid differentiation primary response 88).

12. In Paragraph 1, A composition in which the above extract inhibits the expression of inflammatory proteins.

13. In Paragraph 12, A composition in which the above-mentioned inflammatory protein is PGE2 (prostaglandin E2), iNOS (Inducible Nitric Oxide Synthase), or COX-2 (Cyclooxygenase-2).

14. In Paragraph 1, A composition in which the above extract inhibits the expression of inflammatory cytokines.

15. In Paragraph 14, A composition in which the above inflammatory cytokine is IL-6 or TNF-α (tumor necrosis factor-α).

16. In Paragraph 1, A composition wherein the above-mentioned brain inflammatory disease is a disease selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, neurological autoimmune disease, inflammatory and neuropathic pain, and cerebrovascular disease.

17. A food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

18. A health functional food composition for the prevention or improvement of brain inflammatory diseases comprising an extract of the above-ground parts of *Peucedanum japonicum* as an active ingredient.

19. Step of drying the above-ground parts of the Saposhnikovia divaricata; A step of extracting the above-mentioned dried above-ground parts of *Saposhia divaricata* with a solvent; Method for preparing a food composition for the prevention or improvement of brain inflammatory diseases.

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