KR20230030552A - Composition for immunity enhancement comprising Artemisia gmelinii extract - Google Patents

Abstract

The present invention relates to an immunopotentiation composition comprising an Artemisia gmelinii extract as an active ingredient. The Artemisia gmelinii extract exhibits immunopotentiation effects by proliferating T cells, increasing the production of the cytokines of IL-2 and IFN-γ, increasing the peritoneal macrophage-activating cytokines of IL-6, TNF-α, and IL-12p70, increasing whole blood cells (WBC) which may be the immune cells, neutrophils, lymphocytes, and monocytes, increasing the expression levels of serum IgG and fecal sIgA, and augmenting the activity of natural killer cells to exhibit immunopotentiation effects so as to be advantageously used as an immunopotentiator. In addition, the Artemisia gmelinii extract of the present invention is stable in vivo because of not generating cytotoxicity, and thus can also be utilized even in an immunopotentiation food composition, a pharmaceutical composition, a quasi-drug composition, or a feed composition.

Description

Composition for immunity comprising enhancement Artemisia gmelinii extract}

The present invention is a food composition, health functional food, pharmaceutical composition, quasi-drug composition and feed composition for immunity enhancement containing an extract of heat group ( Artemisia gmelinii ); And it relates to a method for activating NK cells by administering the extract of the hot dog.

Immune response refers to a series of biological reactions that remove substances generated in the body of a living body or materials introduced outside the body when they are different from the living body for the sake of self-integrity, maintenance of individual survival, and continuation of the species.

This immunity is largely divided into innate immunity (innate immunity) and acquired immunity (acquired immunity) obtained by adapting to life. Innate immunity, also called innate immunity, responds non-specifically to antigens and has no special memory function. The innate immune system includes skin and mucous tissues that block the invasion of antigens, strong acidic stomach acid, and complement present in the blood. Cells include macrophages responsible for phagocytosis, polymorphonuclear leukocytes, and K cells that can kill infected cells. Acquired immunity is also called acquired immunity, and it is characterized by being able to remember antigens that invaded for the first time and reacting specifically to effectively remove antigens when invaded again, thereby reinforcing innate immunity.

When this immune function is lowered, various immune diseases such as asthma, seasonal or perennial rhinitis, allergic rhinitis, conjunctivitis, atopic dermatitis, urticaria, hemolysis of red blood cells, acute glomerulonephritis, cold, chronic fatigue and cancer may occur. Therefore, many studies for enhancing immunity have been conducted, and recently, problems with side effects of synthetic compounds have emerged, and thus, development of materials capable of effectively enhancing immunity from natural products is required. For example, a patent has been registered for a “composition for enhancing immunity” containing an extract of Ojeoksan as an active ingredient (Korean Patent Registration No. 10-1141314).

On the other hand, heat keeper ( Artemisia gmelinii ) is a deciduous broad-leaved shrub of the dicotyledonous plant plywood group campanula Asteraceae, and grows in sunny places or fields at the foot of a mountain. The heat keeper has traditionally been used as a diuretic and antipyretic, but there is no report of the immune enhancing activity of the heat keeper.

Against this background, the present inventors have tried to develop a preparation from natural products that exhibits an immunostimulating activity that can prevent and treat 　immune dysfunction while minimizing side effects. has been completed.

The present invention aims to solve the above problems and other problems related thereto.

An exemplary object of the present invention is to provide a composition for enhancing immunity comprising an extract of Artemisia gmelinii as an active ingredient.

Another exemplary object of the present invention is to provide an anti-cancer pharmaceutical composition or anti-cancer adjuvant comprising a deolwigigi extract as an active ingredient.

Another illustrative object of the present invention is to provide a method for activating NK cells by administering a deolwigigi extract.

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 problems mentioned above, and another problem not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object provides a composition for enhancing immunity comprising an extract of Artemisia gmelinii as an active ingredient.

The heat seasoning extract of the present invention proliferates T-cells, increases the production of cytokines IL-2 and IFN-γ, and activates cytokines IL-6, TNF-α and IL-12p70 of peritoneal macrophages. Increase production, increase natural killer cell activity, increase immune cell WBC (Whole Blood Cell), neutrophil, lymphocyte and monocyte production, IgG in Serum, sIgA in Fecal It is characterized by inducing an immune enhancing effect by increasing the expression level.

In the present invention, "heat keeper ( Artemisia gmelinii )" is a deciduous broad-leaved shrub of the dicotyledonous plant plywood group Camphoraceae Asteraceae and is known to commonly grow on the sunny side or field at the foot of a mountain. In oriental medicine, it is known to have effects such as diuresis and antipyretic, and is particularly effective for feverish jaundice. On the other hand, the heat keeper used in the present invention may be purchased and used commercially or harvested or cultivated in nature, but is not particularly limited thereto.

In the present invention, "extract" may mean a product such as a liquid component obtained by immersing a desired substance in a solvent and then extracting it at room temperature or at elevated temperature for a certain period of time, and a solid component obtained by removing the solvent from the liquid component. . In addition, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, purified products, etc. of the results in addition to the results. In addition, a method for extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art.

Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination of two or more methods. In addition, the type of extraction solvent used for extraction of the extract is not particularly limited, and any solvent known in the art may be used. Specifically, the extraction solvent may be at least one selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof.

In the present invention, the extract may be extracted from the stem, leaf, root, outpost or a combination thereof, but is not limited thereto.

In the present invention, "active ingredient" means a component that exhibits the desired activity alone or that can exhibit activity in combination with a carrier that is not active itself.

In the present invention, "immunity" refers to an action or state in which an organism premised on an immune system fights off infection or disease and kills or neutralizes pathogens. It acts as a defense against the invasion of harmful microorganisms. Depending on whether immune cells directly attack a target or attack using proteins or small molecules secreted from immune cells, it can be divided into humoral immunity and cellular immunity. Humoral immunity refers to an immune response made by antibodies produced by B cells, a type of white blood cell. When an antigen invades, B lymphocytes differentiate into plasma cells and memory cells under the influence of helper T lymphocytes, and plasma cells produce antibodies to remove the antigen. Cellular immunity is a concept corresponding to humoral immunity involving antibodies, and refers to an immune process in which cells differentiate between self and non-self and destroy non-self cells. Cell-mediated immunity is divided into an antigen-specific response and an antigen-nonspecific response. Antigen-specific responses are triggered by cytotoxic T cells. Immature cytotoxic T cells differentiate into effector cells that can kill non-self cells when they receive the antigenic determinants handed over by phagocytes, and antigen-specific immune responses occur when antigens bind to the antigen recognition sites of mature cytotoxic T cells. . When cytotoxic T cells and target cells bind, vesicles containing proteolytic enzymes in the cytoplasm of the cytotoxic T cells migrate toward the target cell, and the vesicles are secreted through exocytosis. Among the secreted enzymes, perforin proteins that pierce the cell membrane bind to the cell membrane of the target cell, proteolytic enzymes (granzymes) enter the target cell, and the proteolytic enzyme induces the apoptosis process of the target cell, eventually targeting the target cell. cells die Antigen-specific reactions are caused by natural killer cells (NK cells). NK cells are known to play a key role in destroying virus-infected cells or tumor cells as a primary defense system that acts before an antigen-specific immune response is activated. They work to find and destroy non-self cells without special antigen/antibody reactions.

In the present invention, "immunity enhancement" may mean enhancing biological defense ability against an antigen, and specifically, increasing cellular and humoral immunity against an antigen. The mechanism of immune enhancement is not limited, but may include, for example, promoting the activity of antigen presenting cells such as macrophages or promoting the specific activity of lymphocytes.

The composition of the present invention increases the production of cytokines IFN-γ or IL-2, increases the production of IL-6, TNF-α and IL-12p70, which are cytokines that activate peritoneal macrophages, and immune cells, WBC (Whole Blood Cell), neutrophil, lymphocyte, and monocyte production may be increased, and IgG in Serum and sIgA expression in Fecal may be increased, but is not limited thereto.

In addition, it may be to proliferate T-cells or increase the activity of natural killer cells (NK cells), but is not limited thereto.

The composition of the present invention can be identified as a food composition in a specific embodiment, and the food may include a health functional (sex) food.

In the present invention, the term "health functional food" refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. The above "functionality" means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various forms of dosage form, and unlike general medicines, it has the advantage of using food as a raw material and has no side effects that can occur when taking medicines for a long time, and has excellent portability, so it is free from fine dust. It can be taken as an adjuvant to alleviate cough and phlegm as a respiratory symptom caused by it, and to remove or excrete it.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, and ionic beverages, processed oils such as milk and yogurt, chewing gum, rice cakes, traditional Korean snacks, bread, It can be manufactured into foods such as confectionery and noodles, health functional food formulations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars. In addition, the food composition of the present invention may take any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, health functional foods according to the 'Health Functional Foods Act' of Korea, or snacks, legumes, teas, and beverages according to each food type in the Food Code of Korea's 'Food Sanitation Act' ("Food Standards and Specifications" notified by the Ministry of Food and Drug Safety). , special purpose foods, etc.

In addition, the food composition of the present invention may include food additives in addition to the active ingredient. Food additives may generally be understood as substances that are added to, mixed with, or infiltrated into food in manufacturing, processing, or preserving food. Since food is consumed daily and for a long period of time, its safety must be guaranteed. According to the Food Additives Code under each country's laws governing the manufacture and distribution of food (the 'Food Sanitation Act' in Korea), safety-guaranteed food additives are limitedly regulated in terms of ingredients or functions. In the Korean Food Additives Codex (Ministry of Food and Drug Safety notification 'Standards and Specifications for Food Additives'), food additives are classified into chemical synthetic products, natural additives, and mixed formulations in terms of ingredients, and these food additives are classified as sweeteners and flavors in terms of function It is divided into additives, preservatives, emulsifiers, acidulants, and thickeners.

The sweetener is used to impart an appropriate sweetness to food, and may be natural or synthetic. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

The flavoring agent may be used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and as synthetic flavors, esters, alcohols, aldehydes, terpenes, and the like may be used.

As the preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used, and as the emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin and the like may be used, and acidulant, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like may be used as an acidulant. Acidulants may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to improving taste.

As the thickening agent, a suspending agent, a sedimentation agent, a gel forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and whose stability is guaranteed as food additives for the purpose of supplementing or reinforcing functionality and nutrition. Examples of the physiologically active substance include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine. Minerals include calcium preparations such as calcium citrate and stearic acid. Magnesium preparations, such as magnesium, iron preparations, such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are mentioned.

The food composition of the present invention may contain the above-mentioned food additives in an appropriate amount to achieve the purpose according to the product type, and in relation to other food additives that may be included in the food composition of the present invention, the food code of each country or You can refer to the Food Additives Codex.

In addition, the composition of the present invention can be understood as a pharmaceutical composition in a specific embodiment.

The pharmaceutical composition may be prepared as an oral formulation or parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Specifically, the route of administration may be any appropriate route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, one drug is firstly administered intravenously and the other drug is secondly administered through a topical route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specific reference may be made to the pharmacopoeia of each country including the 'Korean Pharmacopoeia'.

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer according to a method known in the art together with a suitable carrier. It can be prepared in formulations such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol and xylitol, starches such as corn starch, potato starch and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, Serol etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as a lubricant Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. may be mentioned. As disintegrants, starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Moreover, as a diluent, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like can be used.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. These documents are considered as part of this specification.

A preferred dosage of the pharmaceutical composition of the present invention may be determined according to the patient's condition, body weight, sex, age, severity of the patient, and route of administration. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

In addition, the composition of this invention can be grasped as a quasi-drug composition in a specific aspect.

The term "quasi-drugs" of the present invention refers to items that have a milder effect than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, mitigating, treating or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act of the Republic of Korea Quasi-drugs are products excluding items used for pharmaceutical purposes, and include products used for the treatment or prevention of human or animal diseases, products with minor or no direct action on the human body, etc.

The “quasi-drug” composition of the present invention can be prepared in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels or filter fillers, and specifically, disinfectant cleaners, shower foams, wet wipes, solid soaps, liquids It may be soap, hand sanitizer or hand sanitizer, but is not limited thereto.

When the composition for enhancing immunity according to the present invention is used as a quasi-drug or additive, the composition may be added as it is or used together with other quasi-drug or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.

In addition, the composition of this invention can be grasped as a feed composition in a specific aspect.

In the present invention, the term "feed" may refer to any natural or artificial diet, one meal, etc., or a component of the one meal meal, for or suitable for eating, ingesting, and digesting by an animal.

The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

The feed may further include binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and mineral preparations, mineral preparations, protective fatty acid preparations, amino acids, vitamins, enzymes, and probiotics added to increase efficacy (lactic acid bacteria), flavoring agent, non-protein nitrogen compound, silicate agent, buffering agent, coloring agent, extractant, oligosaccharide, etc. .

Another aspect of the present invention for achieving the above object provides an anti-cancer pharmaceutical composition or an anti-cancer adjuvant comprising a deolwigigi extract as an active ingredient.

Another aspect of the present invention for achieving the above object provides a method for activating NK cells by administering a heat group extract.

The heat seasoning extract of the present invention proliferates T-cells, increases the production of cytokines IL-2 and IFN-γ, and activates cytokines IL-6, TNF-α and IL-12p70 of peritoneal macrophages. Increases production of WBC (Whole Blood Cell), Neutrophil, Lymphocyte, and Monocyte, which are immune cells, increases IgG in Serum, sIgA expression in Fecal, and increases natural killer cell activity Since it exhibits an immune enhancing effect, it can be usefully used as an immune enhancing agent. In addition, since the extract of the present invention does not induce cytotoxicity and is stable in the body, it can be used as a composition for food, a pharmaceutical composition, a quasi-drug composition, or a feed composition for enhancing immunity.

However, effects according to one embodiment of the technology disclosed in this specification are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

In order to more fully understand the drawings cited herein, a brief description of each drawing is provided.
Figure 1 shows the results of confirming the cytotoxicity of the heat group ( Artemisia gmelinii ) extract of the present invention.
Figure 2 shows the ratio of T-cells in the splenocytes of mice treated with the extract of heat group.
Figure 3 shows the results of analyzing the degree of IL-2 production according to the heat gigi extract treatment.
Figure 4 shows the results of analyzing the degree of IFN-γ production according to the treatment of the heat group extract.
Figure 5 shows the results of measuring the survival rate of Yac-1 cells according to the treatment of the heat gigi extract.
Figure 6 shows the results of confirming the toxicity in the peritoneal macrophage cells of the heat group ( Artemisia gmelinii ) extract of the present invention.
Figure 7 shows the results of analyzing the degree of IL-6 production according to the treatment of the heat gigi extract.
Figure 8 shows the results of analyzing the degree of TNF-α production according to the treatment of the heat gigi extract.
Figure 9 shows the results of analyzing the degree of IL-12p70 production according to the treatment of the extract of the heat group.
10 shows the results of analyzing the activity of natural killer cells (NK-cell) according to the treatment of the hot air extract.
Figure 11 shows the results of analyzing the degree of WBC (Whole Blood Cell) generation according to the treatment of the extract of the heat machine.
Figure 12 shows the results of analyzing the degree of neutrophil production according to the treatment of the hot air extract.
Figure 13 shows the results of analyzing the degree of monocyte generation according to the treatment of the extract of the hot weather.
Figure 14 shows the results of analyzing the degree of lymphocyte production according to the treatment of the extract of the hot seasoner.
Figure 15 shows the results of analyzing natural killer cell (NK-cell) activity according to the treatment of the hot summer extract in normal mice.
Figure 16 shows the results of analyzing the degree of improvement in IgG in Serum according to the treatment of heat group extract in cyclophosphamide-induced mice.
Figure 17 shows the results of analyzing the degree of improvement in sIgA in Fecal according to the treatment of heat group extract in cyclophosphamide-induced mice.
18 shows the effect of splenocyte proliferation in cyclophosphamide-induced mice according to the heat treatment extract.
Figure 19 shows the effect of splenocyte proliferation in response to contravalin A according to the treatment of the heat group extract in cyclophosphamide-induced mice.
Figure 20 shows the effect of splenocyte proliferation in response to LPS according to the treatment of heat group extract in cyclophosphamide-induced mice.
21 shows the results of analyzing natural killer cell (NK-cell) activity in cyclophosphamide-induced mice treated with heat extract.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited only to these examples.

Preparation Example: Preparation of extracts from hot weather

The heat keeper (extraction site: outpost) samples were cut into 2-5 cm. 50% fermented alcohol was used as an extraction solvent, and the extraction solvent was added to the sample in a weight ratio of 15% to the sample weight. After a large-capacity (stationary) extraction was performed by stirring at 50° C. for 6 hours, the extract was filtered through a filter cotton cloth. The filtrate was then concentrated at 50°C under vacuum conditions and then frozen at -20 to -30°C. For lyophilization, pre-freezing was performed at -38°C for 5 hours or more, followed by freeze-drying at -38 to -10°C for 72 hours. As a result, a powder raw material with a yield of 10 to 12% was obtained.

Example: unicellularization of spleen and culture with extracts from hot weather

The spleen is an important organ for the immune system, and an experiment was conducted to measure the immune activity by culturing the spleen taken from a mouse into single cells. In the case of using splenocytes, compared to measuring the activity of a single cell in a cell line, it can represent an immune response that is relatively similar to the in vivo environment in that a biological organ in which a complex immune response occurs is used.

The spleens were taken from BALB/c mice, converted into single cells, and then used for experiments. For cell culture, RPMI 1640 medium containing 10% Fetal bovine serum (FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin was used. The extracted spleen was ground and mashed, treated with RBC (Red blood cell lysing buffer, Sigma), and the number of cells was measured. The counted cells were treated to a final concentration of 5×10 ⁶ cell/mL using a 48-well cell culture plate, and the heat extract was prepared at a concentration of 12.5 μg/mL or 25 μg/mL at 37° C. and 5% Co-cultivation was performed for 24 hours or 72 hours under conditions of providing carbon dioxide (CO ₂ ).

Cells cultured for 24 hours were collected and used for flow cytometry, and culture supernatants cultured for 72 hours were collected and used for ELISA measurement.

Experimental Example 1: Immune-enhancing activity effect confirmed in lymphocytes of the hot air extract

Experimental Example 1-1: Analysis of cytotoxicity of extracts from hot air

After recovering the culture supernatant prepared by the method of the above example, adding WST-1 reagent to the remaining cells by 1% of the culture medium volume, dispensing 200 μL per well, reacting for 30 minutes, and measuring absorbance at 450 nm using an ELISA reader. measured. The cell viability was expressed as a relative cell viability when the control cells cultured without the sample being treated were 100%.

As a result of co-cultivating the splenocytes and the heat exchanger extract and then treating the WST-1 reagent to measure the absorbance, as shown in FIG. 1, it was confirmed that there was no cytotoxicity at the heat exposure sample concentration used in this experiment.

Experimental Example 1-2: Confirmation of T cell ratio through flow cytometry

Cells cultured in the above example were recovered and flow cytometry experiments were performed. The recovered cells were centrifuged at 1500 rpm for 5 minutes, washed with DPBS, and the number of cells was measured. For each sample, the number of cells was adjusted to 4×10 ⁵ cell/well based on a 96-well plate, and staining was performed for cell surface antigen phenotyping. To prevent non-specific antibody binding, treated with Purified Rat anti-mouse CD16/CD32 (mouse BD Fc Block, BD) and left in the refrigerator for 15 minutes, then cell surface markers CD3 (Brilliant violet 510, Biolegend) and CD19 (Alexa Fluor 488, Biolegend) was suspended in FACS buffer and left at room temperature in a light-shielded state for 30 minutes. The stained cells were washed with PBS and then stained with 7-AAD viability staining solution (Biolegend) for 10 minutes to confirm cell viability. Stained cells were dispensed at 200 μL per well of a 96-well plate, and flow cytometry was measured based on 30,000 dots of live cells in Beckman Coulter's CytoFLEX 3 equipment, and analysis was performed using the CyExpert 2.4 program.

As a result of confirming the effect of the heat keeper extract on immune cells through flow cytometry, as shown in FIG. 2, the ratio of T cells in normal spleen cells (control group) was measured at 21.78%, but the spleen cells treated with the heat keeper extract It was confirmed that the percentage of T cells was increased to 23.87%.

Experimental Example 1-3: Analysis of cytokine producing ability of extracts from hot weather

As in the above example, the culture supernatant cultured for 72 hours was recovered and analyzed for IL-2 and IFN-γ, and the cytokine measurement method was performed according to the protocol of BD, the manufacturer of the ELISA kit.

As a result of measuring the concentration of cytokines in the culture supernatant of the splenocytes co-cultured with the heat exchanger extract, as shown in FIGS. 3 and 4, IL in the supernatant treated with the heat exchanger extract compared to the untreated group (control group) It was confirmed that the concentrations of -2 and IFN-γ were significantly increased.

Experimental Example 1-4: Confirmation of Activation of Natural Killer Cells (NK-cells) by Heat Keeper Extract

In order to confirm the activation of 　immune response in living organisms by the extracts of the hot summer season, a natural killer cell activation experiment, which is widely used to measure the 　immune response activity of drugs, was performed.

Specifically, the spleen was washed with RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and a cell suspension was prepared using a 0.4 μm cell strainer. In addition, spleen cells were obtained after hemolysis of red blood cells with Red blood cell lysing buffer (Sigma-Aldrich Co., St. Louis, MO, USA).

YAC-1 cells were stained using CFSE cell division tracker kit (Biolegend) at a concentration of 1×10 ⁷ cells/ml. Thereafter, the RPMI-1640 culture medium was dispensed in a 96-well plate at a concentration of 4×10 ⁴ cells/well and used as target cells.

Thereafter, the YAC-1 cells, which are target cells, and the separated spleen cells, which are effector cells, were cultured together at a ratio of 1:1, and the extract samples were treated for each concentration and cultured for 24 hours. Cell viability (%) of target cells, CFSE+YAC-1 cells, was measured using a flow cytometer.

As a result, as shown in FIG. 5, it was confirmed that the extract according to the present invention exhibits excellent YAC-1 cell killing ability by activating natural killer cells.

Experimental Example 2: Confirmation of immune enhancing activity effect in macrophages of heat keeper extract

A macrophage activation experiment was performed to confirm the activation of the immune response of organisms by the extracts from the hot summer season.

Specifically, in order to obtain peritoneal macrophages, BALB/c mice were intraperitoneally administered with 2 mL of 3% Brewer thioglycollate medium. After 72 hours, after anesthesia with isoflurane, peritoneal macrophages were obtained using DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

After the obtained peritoneal macrophages were counted, they were treated using a 48-well plate to a concentration of 1x10 ⁶ cells/Ml, and the heat exchanger extract had a concentration of 12.5, 25, and 50 μg/ml at 37°C and 5% Carbon dioxide (CO ₂ ) was incubated for 24 hours under conditions of providing.

The culture supernatant was collected and analyzed for IL-6, TNF-α and IL-12p70, and the cytokine measurement method was performed according to the protocol of BD, the manufacturer of the ELISA kit. In addition, after recovering the supernatant, 500 μL of WST reagent diluted with DMEM, 10% FBS, and 1% Penicillin medium was dispensed per well to the remaining cells, reacted for 30 minutes, and absorbance was measured at 450 nm using an ELISA reader. The cell viability was expressed as a relative cell viability when the control cells cultured without the sample being treated were 100%.

As a result of co-culture of peritoneal macrophages and heat exchanger extract ( Artemisia gmelinii extract, AGE) and treatment with WST reagent to measure the absorbance, as shown in FIG. 6, there is no toxicity at the heat exchanger sample concentration used in this experiment. Confirmed.

In addition, as a result of measuring the concentration of cytokines in the culture supernatant of peritoneal macrophages co-cultured with the heat exchanger extract, as shown in FIGS. 7 to 9, in the supernatant treated with the heat exchanger extract compared to the untreated group (control group) It was confirmed that the concentrations of IL-6, TNF-α and IL-12p70 (FIGS. 7, 8 and 9, respectively) increased significantly.

Experimental Example 3: Immune-enhancing activity confirmed in natural killer cells (NK-cells) of the hot air extract

A natural killer cell activation experiment, which is widely used to measure the immune response activity of a drug, was performed to confirm the activation of the immune response of living organisms by the extract of the hot summer season.

Specifically, spleens were taken from BALB/c mice, converted into single cells, and then used for experiments. For cell culture, RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used. The extracted spleen was ground and mashed, treated with RBC (Red blood cell lysis buffer), and the number of cells was measured. The counted cells were treated to a final concentration of 8 × 10 ⁵ cells/well using a 96-well cell culture plate, and the heat extract was treated to a concentration of 12.5, 25 or 50 μg/mL at 37°C and 5% carbon dioxide. (CO ₂ ) and incubated for 24 hours.

For YAC-1 cell culture, which is the target cell of NK cells in the spleen, RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used at 37℃ and 5% carbon dioxide (CO ₂ ). After culturing and counting under conditions providing , the cells were stained using the CellTrace Violet Cell Prolieferation Kit (Invitogen), and the number of cells was diluted to 2 Х 10 ⁵ cells/mL.

After removing the supernatant after centrifuging the cultured spleen cells, 200 μL of YAC-1 suspension was dispensed and reacted for 4 hours. After centrifugation, the supernatant was removed and 100 μL of Flow Cytometry Staining Buffer (Invitogen) was added. Thereafter, 0.4 μL of 7-AAD was dispensed per well, reacted for 5 minutes, and cell viability (%) of target cells, CTV+YAC-1 cells, was measured using a flow cytometer.

As a result, as shown in FIG. 10, it was confirmed that the extract according to the present invention exhibits excellent YAC-1 cell killing ability by activating natural killer cells.

Experimental Example 4: Confirmation of immune enhancing activity effect in normal mice of heat keeper extract

After orally administering 25 mg/kg of the extract of Dyeoljigi to normal mice, whole blood immune cell analysis and NK natural killer cell (NK-cell) activity were measured.

Experimental Example 4-1: Analysis of immune cells in whole blood

After 2 weeks of oral administration of a heat exchanger extract to a normal mouse, anesthesia with isoflurane, and blood collection through orbital blood collection using a vacuum blood collection tube (BD microtainer_Blood collection tubes, 36594), followed by blood cell analyzer ( HEMAVET 950) was used to measure the number of white blood cells, such as whole blood cells (WBC), neutrophils, lymphocytes, and monocytes.

As a result, as shown in FIGS. 11 to 14, the immune cells in whole blood were significantly WBC (Whole Blood Cell), neutrophil, lymphocyte, and monocyte concentrations in the group ingesting the heat seasoner extract compared to the control group. (Figs. 11, 12, 13 and 14, respectively) were confirmed to have significantly increased.

Experimental Example 4-2: Confirmation of activation of natural killer cells (NK-cell)

After 2 weeks of oral administration of the heat exchanger extract to normal mice, after anesthesia with Isoflurane, the spleen was removed and washed with RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, After unicellularization using a 70 μm cell strainer, it was used in the experiment. The recovered cells were centrifuged (400 × g, 4 minutes, 4 ℃) and treated with RBC (Red blood cell lysing buffer, Sigma) to measure the number of cells. The counted cells were processed to a final concentration of 8×10 ⁵ cells/mL using a 96-well cell culture plate.

YAC-1 cells, target cells of NK cells in the spleen, were stained using the CellTrace Violet Cell Prolieferation Kit (Invitogen), and the number of cells was diluted to 4 Х 10 ⁵ cells/mL.

After removing the supernatant after centrifuging the cultured spleen cells, 100 μL of the YAC-1 suspension was dispensed and reacted for 4 hours. After centrifugation, the supernatant was removed and 100 μL of Flow Cytometry Staining Buffer (Invitogen) was added. Thereafter, 0.4 μL of 7-AAD was dispensed per well, reacted for 5 minutes, and cell viability (%) of target cells, CTV+YAC-1 cells, was measured using a flow cytometer.

As a result, as shown in FIG. 15, it was confirmed that the natural killer cells were activated in the group treated with the extract according to the present invention, resulting in excellent YAC-1 cell killing ability.

Experimental Example 5: Confirmation of immune enhancing activity in mice induced with cyclophosphamide

Cyclophosphamide (CP) 100mg/kg was intraperitoneally administered 3 days prior to the experiment to induce immune suppression after oral administration of 25mg/kg concentration of the extract of heat seasoner to normal mice for 10 days.

Experimental Example 5-1: IgG measurement in Serum

After anesthetizing a normal mouse with isoflurane, blood was collected using a vacuum blood collection tube (BD microtainer_Blood collection tubes, 36594) through orbital blood sampling, and after centrifugation, serum was separated, and the ELISA kit manufacturer The IgG expression level was measured according to the BETHYL protocol.

As a result, as shown in FIG. 16, although the IgG concentration in Serum was significantly decreased by the cyclophosphamide treatment, it was confirmed that the IgG concentration was increased by ingestion of the extract.

Experimental Example 5-2: Measurement of sIgA in Fecal

Fecal 100mg was suspended in 1mL PBS, the suspension was centrifuged (3000 rpm, 10 minutes, 4℃), and the supernatant was diluted 20-fold. Expression of sIgA in feces according to the protocol of MyBioSource, the manufacturer of the Mouse secretory Immunoglobulin A Elisa kit. amount was measured.

As a result, as shown in FIG. 17, it was confirmed that the sIgA concentration in Fecal was significantly decreased by the cyclophosphamide treatment, but the sIgA concentration was increased by the intake of the extract of the hot dog.

Experimental Example 5-3: Measurement of spleen cell proliferation ability

Spleens from BALB/c mice were washed with RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and unicellularized using a 70 μm cell strainer. The recovered cells were lysed with red blood cell lysing buffer (Sigma-Aldrich Co., St. Louis, MO, USA), and then the reaction was terminated and washed to measure the number of cells.

Thereafter, after dispensing 500 μL at a concentration of 5x10 ⁶ cells/mL into a 48-well plate, culturing for 72 hours, removing the supernatant, and adding 500 μL of WST reagent diluted with RPMI 1640, 10% FBS, and 1% Penicillin medium per well. After each aliquot, reaction was performed for 30 minutes at 37°C and 5% of carbon dioxide (CO ₂ ), and then absorbance was measured at 450 nm to measure the proliferative capacity of spleen cells.

As a result, as shown in FIG. 18, it was confirmed that the proliferative capacity of the spleen was significantly increased by ingestion of the extract according to the present invention.

Experimental Example 5-4: Measurement of spleen cell proliferation ability in response to contravalin A and LPS

After dispensing 200 μL at a concentration of 2x10 ⁶ cells/mL in a 48-well plate, the T cell mitogen Contravalin A (Contravalin A, Con A) and LPS were cultured for 48 hours to a final concentration of 1 μg/mL.

Then, the supernatant was removed, and 500 μL of WST reagent diluted with RPMI 1640, 10% FBS, and 1% Penicillin medium was dispensed per well, followed by 30 minutes at 37°C and 5% carbon dioxide (CO ₂ ). After the reaction, the absorbance was measured at 450 nm to measure the proliferative capacity of the spleen cells.

As a result, as shown in FIGS. 19 and 20, it was confirmed that the proliferative capacity of spleen cells corresponding to contravalin A and LPS (FIGS. 19 and 20, respectively) was significantly increased by ingestion of the extract.

Experimental Example 5-5: Confirmation of activation of natural killer cells (NK-cell)

100 μL of spleen cells were dispensed in a 96-well plate to be 8×10 ⁵ cell/well, and then Yac-1 cells, the target cells of NK cells in the spleen, were subjected to CTV staining according to the protocol of Invitogen, the manufacturer of the CellTrace Violet Cell Prolieferation Kit After re-measuring the number of cells, diluting to 4 × 10 ⁵ cells/mL, dispensing 100 μL of Yac-1 suspension, reacting with spleen cells for 4 hours, centrifuging, removing the supernatant, and adding Flow Cytometry Staining Buffer (Invitogen) 100 μL was added. Thereafter, 0.4 μL of 7-AAD was dispensed per well, reacted for 5 minutes, and cell viability (%) of target cells, CTV+YAC-1 cells, was measured using a flow cytometer.

As a result, as shown in FIG. 21, it was confirmed that the natural killer cells were activated in the group treated with the extract according to the present invention, resulting in excellent YAC-1 cell killing ability.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (20)

A food composition for enhancing immunity comprising an extract of Artemisia gmelinii as an active ingredient. According to claim 1,
The extract is water, an alcohol having 1 to 4 carbon atoms, or a mixture of these extracts, the food composition for immunity enhancement. According to claim 1,
The composition increases the production of IFN-γ or IL-2, a food composition for enhancing immunity. According to claim 1,
Wherein the composition increases the production of IL-6, TNF-α or IL-12p70, a food composition for enhancing immunity. According to claim 1,
The composition is to increase the production of WBC (Whole Blood Cell), neutrophil, lymphocyte (Lymphocyte) or monocyte (Monocyte), immune enhancement food composition. According to claim 1,
The composition is to increase the expression level of IgG or sIgA, immune enhancement food composition. According to claim 1,
Wherein the composition proliferates T-cells or increases the activity of natural killer cells (NK cells). A health functional food for enhancing immunity comprising the food composition according to any one of claims 1 to 7. A pharmaceutical composition for enhancing immunity, comprising an extract from the hot air as an active ingredient. According to claim 9,
The extract is extracted with water, alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, a pharmaceutical composition for enhancing immunity. According to claim 9,
The composition increases the production of IFN-γ or IL-2, the pharmaceutical composition for enhancing immunity. According to claim 9,
Wherein the composition increases the production of IL-6, TNF-α or IL-12p70, a pharmaceutical composition for enhancing immunity. According to claim 9,
The composition is to increase the production of WBC (Whole Blood Cell), neutrophil, lymphocyte (Lymphocyte) and monocyte (Monocyte), a pharmaceutical composition for immune enhancement. According to claim 9,
The composition is to increase the expression level of IgG or sIgA, a pharmaceutical composition for immune enhancement. According to claim 9,
Wherein the composition proliferates T-cells or increases the activity of natural killer cells (NK cells), a pharmaceutical composition for enhancing immunity. A quasi-drug composition for immunity enhancement comprising an extract from a hot summer season as an active ingredient. A feed composition for enhancing immunity, comprising an extract from the hot weather as an active ingredient. A pharmaceutical composition for anti-cancer containing an extract from the hot air as an active ingredient. An anti-cancer supplement containing a heat keeper extract as an active ingredient. A method of activating NK cells by administering a heat keeper extract.

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