KR20230139655A - Inflammatory or immune diseases protecting and treating composition comprising ethanol extracts of sea staghorn as active ingredient - Google Patents

Abstract

The present invention relates to a composition comprising an ethanol extract of sea staghorn as an active ingredient for preventing and treating inflammatory or immune diseases. The ethanol extract of sea staghorn according to the present invention has excellent activity in suppressing the production of IL-12 p40, an inflammatory cytokine, in macrophages and dendritic cells caused by stimulating factors that induce inflammatory reactions and outstanding effects of inhibiting the activity of NF-κB, so that the extract can be advantageously used to develop a treatment for inflammatory and immune diseases that can be caused by excessive inflammatory reactions. The ethanol extract of sea staghorn according to the present invention does not cause toxicity to cells to be stable in the body, so that the ethanol extract can be used as a material for functional health food.

Description

Composition for preventing and treating inflammatory or immune diseases comprising auditory ethanol extract as an active ingredient {INFLAMMATORY OR IMMUNE DISEASES PROTECTING AND TREATING COMPOSITION COMPRISING ETHANOL EXTRACTS OF SEA STAGHORN AS ACTIVE INGREDIENT}

The present invention relates to a novel use of auditory ethanol extract that can effectively prevent and treat inflammatory or immune diseases.

Immunity is our body's defense mechanism against pathogens such as bacteria and viruses, and is largely divided into innate immunity, which we have from birth, and adaptive immunity, which we acquire after birth. The innate immune response plays a significant role in protecting against autoimmune diseases such as hyperinflammatory responses (Medzhitov 2001; Lampropoulou et al. 2008). Bone marrow-derived dendritic cells (BMDC) and macrophages (BMDM) are important cellular factors of the innate immune system (Efron et al. 2005), and these cells display pathogen-associated molecular patterns through Toll-like receptors (TLR). Physiology that recognizes (PAMPs) and activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling to trigger an inflammatory response or help with adaptive immunity. Produces active substances (Medzhitov 2001, Takeuchi and Akira 2010). However, when these beneficial substances are excessively produced due to some mutation, autoimmune diseases such as hyperinflammatory reactions occur. Autoimmune disease refers to diseases that occur due to loss of immunological tolerance to self-antigens or an immune response to self-antigens. Meanwhile, at a time when industrial and scientific development is rapidly changing, treatments for diseases such as incurable diseases have been developed, but effective and practical treatments for autoimmune diseases have not yet been developed.

In addition, most of the developed treatments for immune diseases are based on synthetic compounds, which have the problem of causing various side effects in the body and the treatment effect is not very good. Therefore, there is an urgent need to develop new treatments that can effectively treat immune diseases. Accordingly, many researchers have recently shown increasing interest in natural products to develop stable treatments with relatively fewer side effects, and are focusing on developing treatments for immune diseases using natural products. In addition, unlike chemical drugs, natural foods using natural products actually contain many active ingredients that exhibit physiological activities such as antioxidant and anticancer properties in vivo.

In particular, seaweed, which is not only a terrestrial plant but also a marine plant, has greater diversity and novelty than existing terrestrial organisms, and thus has greater potential. Seaweed is largely divided into brown algae, green algae, and red algae. Brown algae include seaweed, kelp, and hijiki. Green algae include Green Algae, Green Algae, and Green Algae, and red algae include Gyeonggi-do, True Red Algae, and Seaweed. Recently, seaweed has received a lot of attention as a health functional food material as it is known to have various physiological activities such as anti-inflammatory, antioxidant, and anti-cancer properties. Codium fagile is a green algae belonging to the Codium fagile family and is distributed along the coasts of Korea, East Asia, and Oceania. Accordingly, while researching new pharmaceutical uses of the auditory ethanol extract, the present inventors discovered that the extract produced IL-12 p40, an inflammatory bioactive substance, in bone marrow-derived dendritic cells and macrophages stimulated with lipopolysaccharides (LPS). It was discovered that it has the activity of suppressing inflammation through the mechanism of inhibiting the breakdown of I┾Bα (inhibitory kappa B-alpha). The present invention was completed by confirming that the auditory ethanol extract extract can be used for the prevention and treatment of inflammatory diseases and immune diseases through this mechanism.

Therefore, the purpose of the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases or immune diseases, which contains as an active ingredient ethanol extract of Acoustic ethanol, which is derived from natural products and has excellent therapeutic effects on inflammatory and immune diseases.

In addition, another object of the present invention is to provide a health functional food for preventing and improving inflammatory diseases or immune diseases containing ethanol extract of Hexaphyllum as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases or immune diseases, containing ethanol extract of Acousticum as an active ingredient.

In one embodiment of the present invention, the auditory extract may be an ethanol extract.

In one embodiment of the present invention, the auditory ethanol extract may be contained in the composition at a concentration of 5 μg/ml to 100 μg/ml.

In one embodiment of the present invention, the ethanol extract may have the activity of inhibiting the production of IL-12 p40, an inflammatory cytokine, and inhibiting the activity of NFκB.

In one embodiment of the present invention, the inflammatory disease may be a systemic inflammatory response caused by microbial infection or sepsis showing symptoms of endotoxic shock.

In one embodiment of the present invention, the immune disease is an autoimmune disease caused by excessive inflammation, such as dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjögren's syndrome. It may be one or more diseases selected from the group consisting of (sjogren's syndrome), multiple sclerosis, and acute and chronic inflammatory diseases.

In addition, the present invention provides a health functional food for preventing and improving inflammatory diseases or immune diseases containing ethanol extract of Hexaphyllum as an active ingredient.

Additionally, the present invention provides a drug for the treatment of inflammatory diseases or immune diseases caused by excessive inflammatory reactions, comprising the composition according to the present invention as an active ingredient.

The auditory ethanol extract according to the present invention has excellent activity in suppressing the production of IL-12 p40, an inflammatory cytokine, in macrophages and dendritic cells caused by stimulating factors that induce inflammatory responses, and has the effect of suppressing the activity of NF-κB. It is excellent and can be usefully used in the development of treatments for inflammatory diseases and immune diseases that can be caused by excessive inflammatory reactions. Since the ethanol extract does not cause toxicity to cells, it is stable in the body and can be used as a material for functional health foods. There is also an effect that can be used.

Figure 1 shows the results of measuring the effect on splenocyte proliferation rate and cytokine secretion ability of the auditory ethanol extract according to the present invention.
Figure 2 shows the results of observing the effect on the proliferation rate of splenocytes in mice ingesting auditory ethanol extract.
Figure 3 shows the results of observing the effect on the cytokine secretion ability of mice ingesting auditory ethanol extract.
Figure 4 shows the results of analysis of the inflammatory cytokine production inhibition activity of the auditory ethanol extract of the present invention.
Figure 5 shows the results of measuring the effect of the auditory ethanol extract of the present invention on inhibiting MAPK activity.
Figure 6 shows the results of measuring the effect of auditory ethanol extract on IκBα degradation.
Figure 7 shows the results of evaluating the effect of the auditory ethanol extract of the present invention on NK cell activity.

The present invention is characterized in that it provides a natural ethanol extract as a new treatment for inflammatory or immune diseases that has excellent anti-inflammatory activity and does not cause intracellular toxicity.

Accordingly, while conducting research to identify new uses for hearing, the present inventors confirmed that auditory ethanol extract has excellent anti-inflammatory activity and can be used as a treatment for inflammatory diseases and immune diseases. According to one embodiment of the present invention, auditory ethanol extract has excellent anti-inflammatory activity. When the extract was treated with bone marrow-derived macrophages and dendritic cells, it was found to very effectively inhibit the production of inflammatory cytokines (eg, IL-12) induced by LPS (see Figure 3).

Therefore, the present invention can provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases or immune diseases containing ethanol extract of Acousticum as an active ingredient. The auditory ethanol extract according to the present invention can be prepared and used by a method known in the art, a modified method thereof, or a method according to the present invention. The auditory ethanol extract of the present invention is produced in a volume amount of about 1 to 30 times the weight of the auditory ear, A solvent selected from water, preferably 5 to 15 times the volume (w/v%) of purified water, lower alcohols with 1 to 4 carbon atoms such as methanol, ethanol, butanol, or mixed solvents thereof, preferably water and methanol. , an ethanol mixed solvent, more preferably 50 to 100% methanol or ethanol, is added and subjected to hydrothermal extraction, ultrasonic extraction, and reflux at about 0 to 100° C., preferably at room temperature, for 10 to 60 hours, preferably 30 to 50 hours. After extraction using an extraction method such as cooling extraction or heating extraction, the extract can be obtained by filtering and concentrating under reduced pressure. In one example of the present invention, an extract was obtained using 80% ethanol.

Meanwhile, the auditory ethanol extract of the present invention obtained by the method described above has the characteristics of having the following functions.

In other words, the auditory ethanol extract of the present invention inhibits the production of IL-12 p40 in bone marrow-derived dendritic cells stimulated by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS), an endotoxic substance, is an inflammatory inducer in dendritic cells and promotes the production of pro-inflammatory cytokines that cause an inflammatory response. In other words, when an external stimulus that can cause an inflammatory response is applied, the expression of inflammatory cytokines such as TNF-α is induced, and the produced inflammatory cytokines stimulate the expression of genes encoding iNOS and COX-2, It causes an inflammatory response by producing NO and PGE2 substances involved in the inflammatory response.

Therefore, if inflammatory substances such as inflammatory cytokines such as TNF-α, IL-6, or IL-12 are secreted excessively or the cells themselves remain activated for a long time, serious side effects such as tissue damage occur.

The auditory ethanol extract according to the present invention has the function of suppressing the production of inflammatory cytokines as described above.

Additionally, the auditory ethanol extract of the present invention inhibits the activity of inflammatory mediators in dendritic cells and macrophages stimulated by lipopolysaccharide (LPS).

Interleukin-12 is a type of interleukin produced by antigen-presenting cells such as dendritic cells and macrophages upon antigen stimulation. It plays a role in stimulating T cells to differentiate them into type 1 helper T cells and NK (natural killer) cells. , plays an important role in the activity of T lymphocytes. It is a heterodimer form of IL-12A(p35) and IL-12B(p40), and the expression of IL-12A(p35) is constitutive, so the biological function of interleukin-12 is mainly IL-12B(p40). It depends on the expression of interleukin-12, and research results have reported that this inhibition of interleukin-12 can be helpful in treating autoimmune diseases, which are excessive immune responses to cells in the body, especially autoimmune diseases mediated by type 1 helper T cells. It is becoming.

Therefore, the auditory ethanol extract of the present invention can be used for the treatment or prevention of inflammatory diseases, which is characterized by acting through the activity of inhibiting the production of TNF-α, IL-6, or IL-12.

In particular, “inflammatory disease” in the present invention is a disease caused by excessive production of inflammatory cytokines TNF-α, IL-6, or IL-12 induced by lipopolysaccharide (LPS), and is an autoimmune disease or sepsis. etc. can be mentioned.

More specifically, the autoimmune diseases include dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, and rheumatic fever. , lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases. Sepsis includes fever symptoms where body temperature rises above 38 degrees, hypothermia where body temperature falls below 36 degrees, respiratory rate increased above 24 beats per minute (tachypnea), heart rate above 90 beats per minute (tachycardia), and blood tests. It includes systemic inflammatory response syndrome (SIRS) and endotoxic shock caused by microbial infection, which shows symptoms of an increase or a significant decrease in the number of white blood cells. More preferably, it is an autoimmune disease, i) Rheumatoid Arthritis, in which the immune system attacks various joint tissues, and ii) autoimmunity of the central nervous system induced by T cells, in which most people can lead relatively normal lives. First, in severe cases, Multiple Sclerosis (MS), which can lead to blindness, paralysis, and premature death; iii) Immunity that occurs when immune cells destroy insulin-producing cells in the pancreas and in which MHC genes play an important role ImmuneMediated or Type Ⅰ Diabetes Mellitus, ⅳ) Inflammatory Bowel Diseases, a disease that occurs when the immune system attacks the intestines, ⅴ) Scleroderma, which induces thickening of the skin or blood vessels (Scleroderma), ⅵ) Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease that is accompanied by symptoms such as deep fatigue, rash, and joint pain. In severe cases, the immune system can cause damage to the kidneys, brain, and lungs. You can. In addition, sepsis includes systemic inflammatory response syndrome (SIRS) and septic (endotoxin) shock, in which a serious inflammatory response occurs throughout the body due to infection with microorganisms. In particular, the main cause of endotoxic shock (septic shock) is excessively produced interleukin-6 and TNF-α.

Therefore, the auditory ethanol extract of the present invention can be used as a pharmaceutical composition for the prevention and treatment of inflammatory diseases, especially sepsis, and autoimmune diseases, especially autoimmune diseases.

Furthermore, the present inventors found that the inhibition of inflammatory cytokine production by the auditory ethanol extract according to the present invention was due to the inhibition of the activity of NF-κB, their upstream signaling system. When PRRs (pattern recognition receptors) recognize and bind to PAMPs, they activate NF-κB signaling and produce inflammation-inducing bioactive substances. Therefore, when NF-κB signaling is inhibited, the production of inflammation-inducing bioactive substances is suppressed, ultimately preventing inflammation and immune diseases.

In one embodiment of the present invention, macrophages treated with the auditory ethanol extract of the present invention and macrophages not treated with the auditory ethanol extract were treated with LPS to induce inflammation, and then the activity of NF-κB and phosphorylation of MAPK were measured. The group treated with the ethanol extract was found to inhibit the activity of NF-κB very effectively compared to the control group (see Figure 3).

For reference, the transcription factor NF-κB (Nuclear factor kappa B) is activated by various external stimuli such as inflammatory cytokines, toxic compounds, bacterial infection, viral infection, radiation, UV, and active oxygen, resulting in cell death and immune response. It is known to regulate the expression of proteins related to various cellular reactions, such as inflammatory reactions. Therefore, abnormal NF-κB activity is associated with various cancers, inflammatory diseases such as arthritis, allergic diseases and inflammatory diseases such as asthma and atopic dermatitis, and substances that inhibit the activity of NF-κB are effective in treating the above diseases. It is becoming a target as a treatment for [A. S. aldwin Jr, J. Clin. Invest., 2001, 107, 241-246].

In addition, NF-κB exists in the cytosol within cells under normal conditions, but when stimulated by free radicals, inflammatory stimuli, carcinogens, toxins, ultraviolet rays, etc., it is activated and moves into the nucleus. It induces the expression of various genes that cause apoptosis inhibition, cell transformation, proliferation, invasion, metastasis or chemical resistance, inflammation, etc. [Chen., Biochem Biophy Res Comm, 2005, 332:1; Jove et al., Endocrinology, 2005, 146:3087; Dandrona et al., Circulation, 2005, 111:1448; Aggarwal et al., Ann N Y Acad Sci, 2004, 1030:434]. Therefore, inhibiting the activity of NF-κB can suppress the expression of these disease-related genes.

In the present invention, the activity of NF-κB was confirmed through the degradation and phosphorylation of IκBα. This means that in the absence of external stimulation, NF-κB forms a complex with an inhibitory protein called IκB (inhibitory-κB) in the cytoplasm. When an extracellular stimulus is given, IκB is inactivated and degraded by phosphorylation, and NF-κB dissociates from IκB and moves to the nucleus, where it becomes active.

In the experimental results of the present invention, it was found that when stimulated by LPS, an external stimulus, the group treated with auditory ethanol extract suppressed the activity of NF-κB by inhibiting the decomposition of IκB compared to the group not treated. .

Meanwhile, the composition according to the present invention may contain a pharmaceutically effective amount of the auditory ethanol extract alone or may include one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, the pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat the symptoms of inflammation or immune disease, and the auditory ethanol extract may be contained at a concentration of 5 μg/ml to 100 μg/ml in the composition. .

Additionally, the pharmaceutically effective amount of the auditory ethanol extract according to the present invention is 0.5 to 100 mg/day/kg of body weight, preferably 0.5 to 10 mg/day/kg of body weight. However, the pharmaceutically effective amount may vary appropriately depending on the degree of symptoms of inflammation or immune disease, the patient's age, weight, health status, gender, administration route, and treatment period.

In addition, 'pharmacologically acceptable' as used herein refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Examples include polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

Additionally, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. Dosage forms may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

The composition according to the present invention can be administered through several routes, including orally, transdermally, subcutaneously, intravenously, or intramuscularly, and the dosage of the active ingredient is determined by various factors such as the route of administration, the patient's age, gender, weight, and patient's severity. It can be appropriately selected depending on. Additionally, the composition for preventing and improving symptoms of inflammatory or immune diseases of the present invention can be administered in combination with known compounds that have the effect of preventing, improving, or treating symptoms of inflammatory or immune diseases.

Accordingly, the present invention can provide a medicament for preventing and treating inflammatory or immune disease symptoms, including a composition containing an ethanol extract of Acoustic acid.

In addition, in the present invention, the inflammatory disease and immune disease are diseases caused by an excessive inflammatory response, and are characterized by the body overreacting to non-specific stimuli due to an uncontrolled inflammatory response. This excessive inflammatory response causes pathological changes that lead to the onset of disease and chronic fixation. For definitions and examples of hyperactive inflammatory disease, see EP0673646.

Furthermore, the auditory ethanol extract according to the present invention does not cause toxicity to cells and does not cause side effects, so it is stable and can be safely used in the body, so it can also be used as a food composition for preventing and improving inflammation or immune diseases. .

Therefore, food compositions for preventing and improving inflammatory or immune diseases containing auditory ethanol extract as an active ingredient are foods that are effective in preventing and improving symptoms of inflammatory and immune diseases, such as main ingredients, secondary ingredients, food additives, and functional foods. Alternatively, it can be easily used as a beverage.

In the present invention, the term “food” refers to a natural product or processed product containing one or more nutrients, preferably in a state that can be eaten directly after a certain degree of processing, and has the usual meaning. This means that it includes all foods, food additives, functional foods, and beverages.

Foods to which the composition for preventing and improving inflammation and immune disease symptoms according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. Additionally, foods in the present invention include special nutritional foods (e.g., infant formula, infant and toddler food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), breads, health supplements, and seasonings. Food (e.g. soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g. snacks), candy, chocolate, gum, ice cream, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, It includes, but is not limited to, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), and natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive can be manufactured by conventional manufacturing methods. In addition, the above-mentioned “functional food” refers to a food group or food composition that provides added value to a food by using physical, biochemical, biotechnological methods, etc. to function and express the function of the food for a specific purpose, such as regulation of biological defense rhythm and disease prevention. It refers to food that has been designed and processed to sufficiently express the body's regulatory functions related to health and recovery, etc., and specifically, it may be a health functional food. The functional food may include food auxiliary additives that are foodologically acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the production of functional foods.

In addition, in the present invention, the term “beverage” refers to a general term for drinks to quench thirst or enjoy taste, and includes functional beverages. The drink has no special restrictions other than containing the composition for preventing and improving the symptoms of inflammation and immune disease as an essential ingredient in the indicated ratio, and like a regular drink, various flavoring agents or natural carbohydrates are added as additional ingredients. It can be contained as.

Furthermore, in addition to those described above, foods containing the composition for preventing and improving inflammatory and immune disease symptoms of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants, and May contain fillers (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 drinks, etc. The components can be used independently or in combination.

In foods containing the composition for preventing and improving inflammatory and immune disease symptoms of the present invention, the amount of the composition according to the present invention may be 0.001% to 90% by weight of the total weight of the food, preferably 0.1%. It may be included in weight% to 40% by weight, and in the case of beverages, it may be contained in a ratio of 0.001g to 2g, preferably 0.01g to 0.1g, based on 100ml, but for health and hygiene purposes or health control. In the case of long-term intake for the purpose, the amount may be below the above range, and the active ingredient can be used in an amount above the above range because there is no problem in terms of safety, so it is not limited to the above range.

Hereinafter, the present invention will be described in more detail through examples. The following examples are merely for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[Example]

Experiment method

Sample preparation

The auditory canal was washed under running water to remove salt. Afterwards, it was dried at 60°C for about 48 hours. The dried perilla was pulverized through a mixer to finally obtain 300 g of persimmon powder. Hearing powder was mixed with 4L of 50% ethanol and soaked and extracted for 48 hours. The extracted extract was concentrated under reduced pressure at 60°C using a reduced pressure concentrator. The ethanol extract that was concentrated under reduced pressure was freeze-dried at -80°C for 72 hours and then used as the final sample.

cell culture

(1) Macrophage culture

RAW264.7 mouse macrophages were purchased from the Korea Cell Line Bank (KCLB, Seoul, Korea) and treated with 100 unit/mL of penicillin and streptomycin, 10% FBS (fetal bovine serum, Gibco BRL, Grand Island, NY, USA) was used after culturing under DMEM (Dulbecco's Modified Eagle's Medium; Gibco BRL, Grand Island, NY, USA) medium at 37°C and 5% CO ₂ conditions.

(2) Mouse preparation

The mice used were C57BL/6 mice purchased from Orient Bio, and they were raised in a sterile place and the night and day periods were set to 12 hours. They were allowed to freely consume solid food and water for rodents and were allowed to acclimatize for one week.

(3) Preparation of mouse bone marrow-derived macrophages and dendritic cells

Bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were obtained from wild-type C57BL/6 mice (Taconic Farm, NY, U.S.A.). Briefly, bone marrow cells were obtained by flushing mouse tibia and femur in Dulbecco's modified Eagles medium (DMEM) medium. The cells were incubated with 10% heat-inactivated FBS (Gibco, NY, U.S.A.) supplemented with 3% J558L hybridoma cell culture supernatant containing granulocyte-macrophage colony-stimulating factor (GM-CSF). , were cultured in RPMI 1640 medium containing 50 μM β-mercaptoethanol and 2 mM glutamine.

In addition, the obtained bone marrow cells were cultured in DMEM medium containing 20% heat-treated FBS, 30% L929 cell culture medium containing macrophage colony-stimulating factor, and 1% penicillin-streptomycin for culturing into macrophages.

(4) Splenocyte isolation

The spleens of mice sacrificed by cervical dislocation were aseptically removed, and RPMI (Roswell Park Memorial Institute) 1640 (RPMI) containing 10% fetal bovine serum and antibiotics penicillin and streptomycin (100 units/mL, 100 μg/mL) was used. After washing three times with (Invitrogen Co., Carlsbad, CA, USA) medium, spleen cells were liberated by homogenization using a tissue grinder (Corning Costar, Corning, NY, USA). To remove red blood cells from the cell suspension, RBC lysis buffer (red blood cell lysis buffer, Invitrogen Co., USA) was added to remove red blood cells, and spleen cells were counted using a hemocytometer.

(5) Measurement of spleen cell proliferation ability

After distributing 1×10 ⁶ spleen cells per well in a 96 well plate, they were divided into groups treated with or without mitogen. The non-mitogen treated group was treated with PBS, and the mitogen treated group was treated with CON. A (concanavalin A) or LPS (lipopolysaccharide) was treated at a concentration of 1 μg/mL, and 24 hours later, 10 μL of WST-1 (Daeil Lap Science, Seoul, Korea) solution was added to each well and incubated for 2 hours. After reaction, absorbance was measured at 450 nm to evaluate cell proliferation ability. Evaluation of cell proliferation ability was expressed as a percentage increase or decrease compared to the control group administered with PBS (phosphate buffered saline, Invitrogen Co., USA) instead of the sample.

(6) Experimental animals and sample administration

Female BALB/c mice (6 weeks old, weighing 15-18 g) were purchased from Orient Bio (Seoul, Korea) and acclimatized for 7 days. The temperature in the breeding room was 23±2℃, relative humidity 50±5%, and light/dark cycle. was kept constant at 12 hours. The animal experiments used in this study were conducted after approval from the Animal Experiment Ethics Committee of the College of Life Sciences, Gangneung-Wonju National University (GWNU 2021-9). After one week of acclimatization, BALB/C mice were divided into 7 groups of 7 mice each. PBS was administered to the control group, and auditory ethanol extract (CFE) was administered at concentrations of 50 and 100 mg/kg body weight (BW). Sample administration for all experimental groups was conducted repeatedly at the same time once a day for 14 days. After the last administration, all groups were fasted for 12 hours and splenectomy was performed. Splenocytes were isolated from the extracted spleen, and spleen cell proliferation ability and cytokine secretion ability were analyzed.

(7) Measurement of NK (Natural killer) cell activity

To isolate NK cells within isolated spleen cells, cells passing through the MS column were negatively selected using MACS (Magnetic activated cell sorting, Miltenyi Biotec, Gladbach, Germany), and the separated NK cells were analyzed using a flow cytometer (Novios, Beckman). Coulter, Miami, FL) was used to confirm that the distribution of CD3-NK1.1 + cells was more than 80%. Using isolated NK cells, the effector cell:target cell (E:T) ratio was 20:1, 10:1, 5:1, and HCT116 cells for cytolytic activity analysis. After reacting 1:1 in a 96 well plate for 18 hours, the lytic ability of the cells was measured at a wavelength of 450 nm using the EZ-LDH kit (Daeil Lap Science, Korea), and the number of target cells used in the experiment was The experiment was conducted by fixing the size at 4.5×10 ⁴ .

(8) Measurement of cell viability by auditory ethanol extract

To check cytotoxicity, RAW264.7 cells were cultured in a 96-well plate at a concentration of 2×10 ⁴ cells/well for 24 hours. LPS was dissolved in phosphate buffered saline (PBS) at a concentration of 100 ug/ml, diluted to a concentration of 1, 2.5, and 5 ug/ml in each well, and treated at a volume of 10 ul for 24 hours. The control group was treated with 10 ul of PBS for 24 hours. After drug treatment was completed, 10ul of EZ-cytox reagent was added to each well and cultured in an environment of 37°C and 5% CO ₂ for 2 hours. Absorbance was measured at 450 nm using SYNERGY HTX (Biotek) equipment.

(9) Effect of auditory ethanol extract on changes in cellular protein expression (Westerblotting)

Cells were cultured at 8×10 ⁵ cells/well in a 60 mm plate for 24 hours. Auditory ethanol extract (CFE) was diluted in DMSO to a concentration of 20 mg/ml, diluted to a concentration of 5 μg/ml using cell culture medium, and pretreated for 1 hour. LPS was treated at a concentration of 1ug/ml on each plate for 15, 30, and 60 minutes, respectively. After drug treatment, the cells were washed once with cold PBS and collected in a 15ml tube using Trypsin EDTA. The collected cells were centrifuged at 1500 RPM for 5 minutes, the supernatant was removed, resuspended using PBS, and centrifuged at 1500 RPM for 5 minutes. The washed cells were lysed using 100 μl of 2X SDS-Sample buffer. Samples with completed lysis were boiled at 95°C for 5 minutes. The BCA A and BCA B solutions were diluted at a ratio of 50:1 and 98 μl each was added to a 96-well plate. Then, 2 μl of the protein sample was added to each well and reacted at 60°C for 5 minutes. And the degree of cell death was analyzed. Absorbance was quantified by measuring absorbance at 562 nm using SYNERGY HTX (Biotek) equipment. Proteins were separated through SDS-PAGE, and transfer was performed using a PVDF membrane. The membrane with completed transfer was blocked using 5% skim milk (in TBST) at room temperature for 1 hour. The primary antibody was reacted with 5% BSA (in TBST) (beta actin, alpha tublin, Cleaved Caspase3, Cleaved PARP, SOD2, Catalase) at 4°C for 12 hours. The secondary antibody was reacted at room temperature for 1 hour using 5% skim milk (in TBST).

(10) ROS scavenging effect of auditory ethanol extract (CFE) on macrophages

Cells were cultured in a 6-well plate at a concentration of 6×10 ⁵ cells/well for 24 hours. Auditory ethanol extract (CFE) was diluted in DMSO to a concentration of 20 mg/ml, diluted to a concentration of 5ug/ml using cell culture medium, and pretreated for 1 hour. LPS was treated at a concentration of 1ug/ml in each well for 1 hour. RAW264.7 cells that had completed drug treatment were washed twice with PBS. Cells were collected using 0.05% Trypsin EDTA and centrifuged at 1500 RPM for 3 minutes. Afterwards, the cells were resuspended using PBS and the cells were transferred to a 1.5ml tube. After centrifugation at 1500 RPM for 3 minutes, cells were resuspended using phenol red free HBSS. Each cell was treated with CMH2DCFDA at a concentration of 5 μM and reacted at 37°C for 30 minutes. At the end of the reaction, the ROS scavenging ability was measured immediately using CYTOFLEX (BECKMAN) equipment.

(11) Statistical processing

Significance was assessed using the ANOVA test using the GraphPad Prism (GraphPad Software, USA) program. Data were expressed as standard error of the mean (SEM). Significance was evaluated as *p<0.05, **p<0.01, ***p<0.001.

Experiment result

1. Evaluation of the effect of auditory ethanol extract on splenocyte proliferation rate and cytokine secretion ability

In the body, the spleen is an organ responsible for the main protective immune response to antigens derived from the blood. It is a major lymphoid organ where maturation of B and T lymphocytes and differentiation of lymphocytes by stimulation of antigens occur. Therefore, proliferation of spleen cells is very important in the immune system. In particular, spleen cells are closely related to the immune response, and their size and number can be used as direct indicators, so they are used as a representative immune indicator. Therefore, in order to observe the effect of the auditory ethanol extract on the splenocyte proliferation rate and cytokine secretion ability, the spleen was removed from BALB/C mice and the freed splenocytes were treated with the auditory ethanol extract (CFE) of the example, respectively, to determine the splenocyte proliferation rate and cytokine secretion ability. Cytokine secretion ability was measured, and the results are shown in Figure 1.

As a result of treating the auditory ethanol extract at different concentrations (5 μg/ml, 10 μg/ml, 30 μg/ml, and 50 μg/ml), no cytotoxicity was observed in all extract treatments, and the proliferative ability of splenocytes in the treatment groups was low. It appeared to increase significantly (P < 0.05) (see A in Figure 1). In addition, as a result of observing the secretion ability of cytokines (IL-2 and IFN-γ), similar to the results of spleen cell proliferation ability, the secretion ability of IL-2 and IFN-γ in the CFE treatment group was significantly dependent on the treatment concentration (P < 0.05, P < 0.01, P < 0.001) was found to increase (see B, C of Figure 1).

2. Splenocyte proliferation rate and cytokine secretion capacity of mice ingesting auditory ethanol extract

In order to observe the effect of administration of the ethanol extract on the immune system of mice, the ethanol extract was administered at 50 or 100 mg/kg BW once a day for 14 days, and the spleens of the administered mice were removed and extracted from the spleen. The proliferation ability and cytokine secretion ability of the derived spleen cells were observed. Mitogen is a general term for substances that stimulate specific immune cells and promote their activation. These mitogens exist in various ways depending on the type of cell. Typical mitogens used in spleen cell experiments include CON A (concanavalin A) and LPS (lipopolysaccharide). CON A stimulates T cells, induces cell proliferation and increases secretion of cytokines, and LPS stimulates T cells. Stimulates cells and induces cell activity. Therefore, this combination treatment of mitogens can increase the sensitivity of cells to sample processing, making it easier to determine whether cells are active. As a result of examining the proliferative ability of splenocytes isolated from mice administered auditory ethanol, the proliferative ability of splenocytes was found to be significantly increased in the auditory ethanol-treated group in the mitogen-free group, mitogen CON A-treated group, and mitogen LPS-treated group, and in the auditory ethanol-treated group. No significant changes in spleen cell proliferation ability were observed (see Figure 2).

In addition, as a result of observing the secretion ability of cytokines in the spleen cell culture supernatant, similar to the results of the proliferation ability of spleen cells, the secretion of IL-2 and IFN-γ was significantly higher in the mitogen-free group and the mitogen CON A treatment group than in the auditory ethanol-treated group. appeared to increase (Figure 3 A, B). As a result of the above results, it was confirmed that auditory ethanol extract has an immune-enhancing effect through in vitro and in vivo experiments.

3. Analysis of inflammatory cytokine production inhibition activity of auditory ethanol extract

In order to confirm whether the auditory ethanol extract has the activity of suppressing the production of inflammatory cytokines that cause inflammation, the following experiment was performed. Macrophages and dendritic cells derived from the previously prepared mouse bone marrow were distributed in a ⁴⁸ -well plate at a cell count of 1 It was treated at each concentration of 50μg/ml. At this time, a group not treated with auditory ethanol extract was used as the control group. 18 hours after treatment with 10 ng/ml of LPS, cell culture was obtained, and the expression level of IL-12 p40 was measured using ELISA (BD PharMingen, CA, USA, R&D system, MN, USA).

As a result, as shown in Figure 4, in the case of macrophages stimulated with LPS without treatment with the auditory ethanol extract, the production of IL-12 p40, an inflammatory cytokine, was found to be significantly increased, whereas the auditory ethanol extract of the present invention showed a significant increase. The group treated showed that the production of inflammatory cytokines was significantly inhibited in proportion to the treatment concentration.

Through these results, it was found that the auditory ethanol extract has the activity of effectively suppressing inflammatory cytokines that occur as inflammation-inducing factors, and therefore, it can be used as a preventive and therapeutic agent for diseases that may be caused by inflammation. I was able to.

4. Analysis of MAPK activity inhibition effect of auditory ethanol extract

As the results of 1 above confirmed that the auditory ethanol extract has the activity of suppressing the production of inflammatory cytokines induced by LPS, it was confirmed that the auditory ethanol extract can regulate the activity of MAPK, an upper signaling system that generates inflammatory cytokines. This was confirmed through Western blot. For this purpose, first, macrophages were distributed in a ⁶⁰ -mm dish at a cell count of 5 Processed. At this time, a group not treated with auditory ethanol extract was used as the control group. Afterwards, cells were collected and lysed using elution buffer (PRO-PREP Lysis buffer, iNtRON Biotechnilogy), and protein samples were electrophoresed in 10% SDS PAGE, then electrically transferred to a polyvinylidene fluoride membrane, and then 1/ After treatment with primary antibodies diluted 1000, phospho-p44/42 (P-ERK1/2), phospho-p38, p38 MAPK, phospho-JNK1/2 (Cell Signaling Technology, MA, USA) antibodies were used for reaction. , the membrane was washed and reacted again with horseradish peroxidase-linked anti-rabbit IgG (Cell Signaling Technology), and then the signal was confirmed using the WEST-ZOL plus western blot detection system (iNtRON Biotechnology).

As a result of Western analysis, as shown in Figure 5, when treated with auditory ethanol extract, there was a significant inhibitory effect on phosphorylated ERK1/2 and JNK1/2, while there was no change in the amount of phosphorylation of p38 (see Figure 5). Therefore, it was confirmed that the extract according to the present invention has the effect of inhibiting the phosphorylation of MAP kinases (ERK and JNK).

5. Analysis of the effect of auditory ethanol extract on IκBα degradation

TLR4 stimulation by LPS induces phosphorylation of IκB by IκB kinase and ubiquitination and degradation of IκB followed by activation of NF-κB (Bao L, et al., Brain Pathol. 12: 420-429 (2002 )). Therefore, activation of NF-κB was analyzed indirectly by degradation of IκBα. Figure 6 shows that auditory ethanol extract has an inhibitory effect on the degradation of IκBα. A portion of bone marrow-derived macrophages were treated with 50 μg/ml of ethanol extract, and the remaining portion was not treated. After 1 hour, both extract-treated and untreated cells were stimulated with 10 ng/ml of LPS. Total cell lysates are obtained at each time interval, and SDS-PAGE and immunoblotting are performed to estimate the degradation of IκBα. Total p38 is used as a loading control.

As can be seen in Figure 6, as a result of immunoblotting, IκBα was decomposed by LPS stimulation and the amount was small, but after treatment with the auditory ethanol extract, the decomposition was suppressed and the amount increased compared to before treatment. Therefore, it was found that the extract according to the present invention has the effect of inhibiting the decomposition of IκBα and thus inhibits the activation of NF-κB.

6. Assessing the effect on NK cell activity

In order to evaluate the NK cell activation ability of the ethanol extract of the ethanol extract through spleen cell proliferation and cytokine secretion ability, the ethanol extract of the ethanol extract was administered at 100 mg/kg BW once a day for 14 days, and the spleens of the administered mice were removed and spleen Mature NK cells within the cells were isolated, and cancer cells (Target cells; HCT116) were mixed with the separated NK cells (Effector cells) to evaluate NK cell activity.

As a result of measuring the activity of NK cells to kill cancer cells by treating NK cells isolated from the spleen with cancer cells at different ratios (1:1, 5:1, 10:1, 20:1), the effector cells were affected in a concentration-dependent manner. NK cell activation appeared to increase (Figure 7).

The highest NK cell activation capacity was observed at a ratio of Effector cell:Target of 20:1, and when comparing the efficacy between the control group and the auditory ethanol extract administration group, all ratio treatments (1:1, 5:1, 10:1, 20:1), the NK cell activation capacity of the ethanol extract was observed to be significantly higher.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (4)

A pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising an ethanol extract as an active ingredient. According to clause 1,
The auditory ethanol extract is a pharmaceutical composition for preventing or treating autoimmune diseases, characterized in that it is an ethanol extract. According to clause 1,
A pharmaceutical composition for the prevention or treatment of autoimmune diseases, characterized in that the auditory ethanol extract is contained in the composition at a concentration of 5 μg/ml to 100 μg/ml. According to clause 1,
A pharmaceutical composition for the prevention or treatment of autoimmune diseases, wherein the ethanol extract has the activity of inhibiting the production of IL-12 p40, an inflammatory cytokine, and inhibiting the activity of NF-κB.