KR20240083416A - A composition for reinforcing immune function comprising herbal mixture extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing immunity containing mixed extracts of Astragalus, Angelica root, and Chrysanthemum pollen as active ingredients, and a method for enhancing immunity using the pharmaceutical composition.

Description

A composition for reinforcing immune function comprising herbal mixture extract}

The present invention relates to a pharmaceutical composition for enhancing immunity containing mixed extracts of Astragalus, Angelica root, and Chrysanthemum pollen as active ingredients, and a method for enhancing immunity using the pharmaceutical composition.

Immune function regulation is a term that comprehensively encompasses the action of restoring normal immune function or alleviating changes in the body's normal immune function caused by environmental pollutants, side effects of medicines, diseases, and aging, and is generally undesirable. It is classified into immunosuppressive action, which is a regulatory action that suppresses an increased immune response, and immune enhancing action, a regulatory action that increases a decreased immune response.

Among these, immunostimulation is one of the important therapeutic strategies that strengthens the body's defense mechanism against various diseases such as cancer and inflammatory diseases. It stimulates the immune response by increasing the activity of immune cells to achieve an immune enhancement effect. You can. For example, macrophages play a major role in the immune response. Phagocytosis, a major role of macrophages, absorbs microorganisms and other pyrogenic particles, and also produces tumor necrosis factor-α (TNF-α) and interleukin. -Immune response by secreting a number of cytokines such as interleukin (IL-1β) and interleukin-12 (IL-12) and cytotoxic and inflammatory substances such as nitric oxide (NO) (Wolf et al., 1994; Lee and Hong, 2011; Murray and Wynn, 2011). Therefore, increasing macrophage activity can be a means to enhance immunity. Since infections and diseases mainly occur when immune function is reduced, various studies are being conducted on how to enhance immune responses through immune-enhancing substances when the body's immune system function is reduced.

Meanwhile, macrophages are known to be involved in host defense and homeostasis by participating in various host responses such as innate immunity as well as adaptive immunity, and during immune responses, IL-1β (interleukin-1β) and IL-6 ( It is known as a cell that plays an important role in biological defense in the early stages of infection by producing cytokines such as interleukin-6) and tumor necrosis factor-α (TNF-α) (Journal of Pharmaceutical Sciences 2013; 57(6): 394 ~399).

Against this background, the present inventors have made extensive research efforts to develop a preparation that can safely and effectively enhance immunity. As a result, they have confirmed that a mixed herbal extract of Astragalus, Angelica root, and Chrysanthemum pollen can effectively enhance immunity, and completed the present invention. did.

One object of the present invention is to provide a pharmaceutical composition for enhancing immunity containing a mixed extract of Astragalus, Angelica root, and Astragalus root as an active ingredient.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract of Astragalus, Angelica gigas, and Chrysanthemum pollen contains granulocyte colony formation promoting factors in macrophages, IL-12, IL-2, IL-6, It may indicate the ability to promote TNF-α or NO production.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen has the ability to promote proliferation in spleen cells, the ability to promote NK cell activity, IFN-γ, IL-12, It may indicate the ability to promote IL-2, TNF-α or NO production.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract is a mixture of astragalus, angelica root, and perennial pollen, mixed with one selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. It may have been extracted with more than one type of solvent.

In one of the above-described embodiments, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v/v) ethanol.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract of Astragalus, Angelica root, and Astragalus root may be mixed at a weight ratio of Astragalus: Angelica root: Angelica root = 0.5 to 5:1:1. .

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the composition may further include a pharmaceutically acceptable carrier, excipient, or diluent.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the composition is administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, or intranasally. , may be administered through intrapulmonary, or intrarectal administration routes.

Another object of the present invention is to provide a method for enhancing immunity comprising administering the composition to an entity other than a human.

Another object of the present invention is to provide a food composition for enhancing immunity containing a mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases containing a mixed extract of Astragalus, Angelica root, and Astragalus root as an active ingredient.

In the pharmaceutical composition for preventing or treating immune diseases of the present invention, in one embodiment, the immune diseases include cold, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergy, rheumatoid arthritis, and Alzheimer's disease. The disease may be selected from the group consisting of autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, and psoriasis.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

One aspect of the present invention is to provide a pharmaceutical composition for enhancing immunity containing a mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen as an active ingredient.

The term "Astragalus membranaceus" in the present invention refers to a plant referred to by the scientific name ' Astragalus membranaceus '; “Angelica” is a plant referred to by the scientific name ‘ Angelica gigas ’; and "Thousand Pollen" respectively refer to plants referred to by the scientific name ' Trichosanthes kirilowii Maximowicz'.

Astragalus membranaceus is a perennial plant of the dicotyledonous Rosaceae legume family and is distributed in Korea, Japan, Manchuria, northeastern China, and eastern Siberia. It is commonly cultivated as a medicinal herb. In oriental medicine, it is collected in the fall, removes the outcrops and fine roots, and dries it in the sun. It is called astragalus of herbal medicine. It is known to have better medicinal effects if used as is without peeling. It has tonic, antiperspirant, diuretic, and analgesic effects, so it is prescribed for physical weakness, fatigue, boredom, and cold sweat.

Angelica gigas is the dried root of Angelica gigas, a perennial herb belonging to the Apiaceae family. It has a sweet, very sweet taste and warm properties. The efficacy of angelica root includes a blood-replenishing effect that produces blood when blood is insufficient. Angelica root promotes blood flow in the coronary arteries and stimulates the production of red blood cells. In addition, it has been reported that angelica root extract containing decursin and/or decursinol angelate can be used as an anticancer composition (Korean Patent No. 10-1245328).

Trichosanthes kirilowii Maximowicz refers to the peeled roots of the perennial Hanultari or Yellow Hanultari belonging to the Cucurbitaceae family. It has no smell, tastes bitter and sour, and is cold in nature. Treats dryness, boils, and pus when the essence is damaged by heat. It mainly reduces fever in the lungs and stomach and produces a fluid to quench thirst and moisturize the body.

In the present invention, the ethanol extracts of Astragalus, Angelica root, and Astragalus chinensis can be used interchangeably with "SH003" in the same sense.

In the present invention, the astragalus, angelica root, or pollen can be purchased and used commercially, or those collected or cultivated in nature can be used, but are not limited thereto. Additionally, the extract of Astragalus, Angelica gigas, or Chinese pollen can be extracted from natural, hybrid, or mutated plants, or from plant tissue culture.

The term "extract" in the present invention refers to an extract obtained by extracting and processing a mixture of Astragalus, Angelica root, and Chinese pollen, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, and the extract of the extract. It includes the extract itself, such as fractions or mixtures thereof, and all formulations of the extract that can be formed using the extract.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract is a mixture of astragalus, angelica root, and perennial pollen, mixed with one selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. It may be extracted with one or more solvents, specifically ethanol extract, but is not limited thereto.

In any one of the above-mentioned embodiments, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v/v) ethanol, specifically 30% (v/v) ethanol, but is limited thereto. It doesn't work.

In the mixed extract of astragalus, angelica root, and perennial pollen of the present invention, the method of extracting the mixture 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 hot water extraction, ultrasonic extraction, filtration, and reflux extraction, which may be performed alone or by combining two or more methods.

Additionally, the extract may be prepared and used in dry powder form after extraction, but this is not limited.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract of Astragalus, Angelica root, and Astragalus root powder is in a weight ratio of Astragalus: Angelica root: Angelica root = 0.5 to 5:1:1, specifically, Astragalus: It may be mixed at a weight ratio of Angelica: Angelica root = 1 to 3:1:1, more specifically, Astragalus: Angelica root: Thousand pollen = 1:1:1, but is not limited thereto.

In the present invention, the term "immunity enhancement" refers to enhancing biological defense ability against antigens, and may specifically mean increasing cellular and humoral immunity against antigens.　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.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract of Astragalus, Angelica gigas, and Chrysanthemum pollen contains granulocyte colony formation promoting factors in macrophages, IL-12, IL-2, IL-6, It may indicate the ability to promote TNF-α or NO production.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen has the ability to promote proliferation in spleen cells, the ability to promote NK cell activity, IFN-γ, IL-12, It may indicate the ability to promote IL-2, TNF-α or NO production.

The pharmaceutical composition for enhancing immunity of the present invention can improve immune suppression through activation of macrophages, spleen cells, and NK cells.

The active ingredient of the mixed extract of Astragalus, Angelica root, and Astragalus chinensis is not particularly limited thereto, but may be nodakenin or the like. The nodakenin can be isolated from natural sources, such as mixed extracts of Astragalus, Angelica root, and Astragalus root, but is not limited thereto, and may be chemically synthesized by a method known in the art, or may be a commercially available material.

The pharmaceutical composition of the present invention may contain 0.001 to 80% by weight of the extract, specifically 0.001 to 70%, and more specifically 0.001 to 60% by weight based on the total weight of the composition, but is not limited thereto.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the composition may further include a pharmaceutically acceptable carrier, excipient, or diluent, but is not limited thereto.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the preparation of the pharmaceutical composition, and the carrier may include a non-naturally occurring carrier. there is.

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

Specifically, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. can be used In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium phosphate. , calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, or lactose. It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups, and may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to the commonly used simple diluents such as water and liquid paraffin. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogenatin, etc. can be used.

In the pharmaceutical composition for enhancing immunity of the present invention, in one embodiment, the composition is administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, or intranasally. , may be administered through intrapulmonary or intrarectal administration routes, but is not limited thereto.

Another aspect of the present invention provides a method for enhancing immunity comprising administering the composition to an entity other than a human.

The term "administration" in the present invention means introducing a predetermined substance into an individual by an appropriate method.

The term "individual" in the present invention refers to all animals such as rats, mice, and livestock, including humans, that require immunity enhancement. Specific examples include, but are not limited to, mammals, including humans.

Specifically, the method for enhancing immunity of the present invention may include the step of administering a pharmaceutical composition containing a mixed extract of Astragalus, Angelica root, and Astragalus root as an active ingredient to an individual other than a human in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" of the present invention refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's gender, Factors including age, weight, health status, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs combined or used simultaneously, and other medical factors. It can be easily determined by a person skilled in the art according to factors well known in the art.

The pharmaceutical composition may be administered alone or in combination with other immune-enhancing agents, and may be administered sequentially or simultaneously with conventional immune-enhancing agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

For example, the composition of the present invention can be administered at 0.0001 to 100 mg/kg of body weight per day, more specifically 0.001 to 100 mg/kg of body weight, based on solid content. The recommended dosage may be administered once a day, or it may be administered several times.

In addition, the administration route and method of administering the composition are not particularly limited, and any administration route and method may be used as long as the composition containing the composition can reach the desired area. Specifically, the composition may be administered through various oral or parenteral routes, and non-limiting examples of the administration route include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, and nasal. It can be administered internally or through inhalation, and the dosage varies depending on the patient's condition and weight, degree of disease, drug type, administration route, and time, but can be appropriately selected by a person skilled in the art.

Another aspect of the present invention provides a food composition for enhancing immunity containing a mixed extract of Astragalus, Angelica root, and Astragalus root as an active ingredient.

As used herein, “food” refers to meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcoholic beverages. , vitamin complexes, health functional foods, and health foods, etc., and include all foods in the conventional sense.

The food composition of the present invention can be expected to have a high immune-enhancing effect because it is derived from a mixed extract of astragalus, angelica root, and perennial pollen, which can be consumed on a daily basis, and can therefore be very useful for health promotion purposes.

The above-mentioned health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, with high medical effects. It means food. Here, ‘function’ means controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food.

The food composition of the present invention can be manufactured in various types of formulations, and unlike general drugs, it is made from natural products and has the advantage of not having side effects that may occur when taking the drug for a long period of time, and is excellent in portability, so the present invention Foods can be consumed as supplements to improve immunity.

The above-mentioned health food refers to food that has a more active health maintenance or promotion effect compared to general food, and health supplement food refers to food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement are used interchangeably.

Specifically, the health functional food is a food manufactured by adding the compound of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspending it, and consuming it may cause certain health problems. It means bringing about an effect, but unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art can be used.

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

In addition, the food composition contains preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxide) roxitoluene (BHT), etc.), colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG monosodium glutamate, etc.), sweeteners (dulcine, cyclemate, saccharin) , sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), strengtheners, emulsifiers, thickeners (grease), coating agents, gum base agents, anti-foam agents, solvents, improvers, etc. May contain food additives. The additives can be selected depending on the type of food and used in an appropriate amount.

As an example of the food composition of the present invention, it can be used as a health drink composition, and in this case, it can contain various flavoring agents or natural carbohydrates as additional ingredients, like ordinary drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g, per 100 ml of the health drink composition of the present invention.

In addition to the above, the health drink composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or carbonating agent. Additionally, it may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating immune diseases comprising a mixed extract of Astragalus, Angelica root, and Astragalus root as an active ingredient.

The term "prevention" of the present invention refers to any act of suppressing or delaying an immune disease by administering an immune-enhancing pharmaceutical composition containing the mixed extract of Astragalus, Angelica root, and Astragalus chinensis as an active ingredient.

The term “treatment” of the present invention refers to any action in which the symptoms of an individual suspected or affected by an immune disease are improved or beneficially changed by administration of the pharmaceutical composition.

The term "immune disease" of the present invention includes various inflammatory diseases, allergic diseases, and cancer caused by abnormalities in the body's immune system, such as cold, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, It may include, but is not limited to, atopic dermatitis, allergies, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, and psoriasis.

The composition of the present invention may contain one or more known active ingredients that have the effect of enhancing immunity or preventing and treating immune diseases, along with mixed extracts of Astragalus, Angelica root, and Chrysanthemum pollen.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response regulators to enhance immunity or prevent and treat immune diseases.

The mixed extract of Astragalus, Angelica root, and Chinese pollen of the present invention can exhibit an activating effect on macrophages, spleen cells, and NK cells, and can therefore be usefully used as an immune enhancer and immune stimulant for various diseases caused or worsened by immunodeficiency. there is.

Figure 1 is a diagram showing the results of detecting nodakenin by HPLC analysis in the mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen of the present invention.
Figure 2 is a diagram showing the results of analyzing the effect of improving the macrophage immunostimulatory activity of the mixed extract of Astragalus, Angelica root, and Astragalus root of the present invention or Nodakenin.
Figure 3 is a diagram showing the results of analyzing NO production in macrophages of the mixed extract of Astragalus, Angelica root, and Astragalus root of the present invention or Nodakenin.
Figure 4 is a diagram showing the results of analyzing the NF-κB activation effect on macrophages of nodakenin, a mixed extract of astragalus, angelica root, and perennial pollen of the present invention.
Figure 5 is a diagram showing the results of analyzing the effect of improving the spleen cell immunostimulatory activity of the mixed extract of Astragalus, Angelica root, and Astragalus root of the present invention or Nodakenin.
Figure 6 is a diagram showing the results of analyzing NO production in spleen cells of the mixed extract of Astragalus, Angelica root, and Astragalus root of the present invention or Nodakenin.
Figure 7 is a diagram showing the results of analyzing the effect of the mixed extract of Astragalus, Angelica root, and Astragalus root of the present invention or Nodakenin on promoting proliferation of spleen lymphocytes.
Figure 8 is a diagram showing the results of analyzing the effect of the mixed extract of astragalus, angelica root and perennial pollen of the present invention or nodakenin on promoting NK cell activity in spleen cells.
Figure 9 is a diagram illustrating the effect of the mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen of the present invention or nodakenin in alleviating CP-induced immune suppression in a mouse model of CP-induced immune suppression.
Figure 10 is a diagram illustrating the effect of the mixed extract of Astragalus, Angelica root, and Astragalus root of the present invention or nodakenin in alleviating CP-induced immune suppression in the spleen of a mouse model of CP-induced immune suppression.

Hereinafter, to aid understanding of the present invention, it will be described in detail through examples. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Example 1. Preparation of fractions and nodakenin of mixed herbal ethanol extract (SH003)

Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes kirilowii Maximowicz (Tk) were mixed at a weight ratio (w/w) of 1:1:1, respectively, placed in an extractor, and 30% ( v/v) extracted with ethanol at 100°C for 3 hours. The extract was filtered, the filtrate was concentrated under reduced pressure, and then dried to obtain a mixed extract of Astragalus, Angelica gigas, and Astragalus root. The dried extract was dissolved in 30% ethanol and stored at -80°C until use. When used, it was again dissolved in 30% ethanol to prepare a concentration of 100 mg/ml, and then further diluted with culture medium.

Nodakenin (purity ≥97%, Ensol Biosciences Inc., Daejeon, South Korea) was prepared according to a previous study (Li et al., 2020). When used, nodakenin was dissolved in dimethyl sulfoxide (DMSO) and further diluted with culture medium (DMSO <0.2%).

Example 2. Analysis of Nodakenin

To standardize the SH003 extract for the presence of Nodakenin, Nodakenin in SH003 was analyzed by an HPLC system (Shimadzu 20A, Shimadzu Co., Kyoto, Japan). Analysis was performed on a Shim-pack GIS-5 μm ODS column (250 mm × 4.6 mm; column temperature, 30 °C; injection volume, 10 μl). The mobile phase consisted of (A) distilled water and (B) acetonitrile at a flow rate of 1.0 ml/min. The results are shown in Figure 1.

As shown in Figure 1, nodakenin was confirmed to be detected at a retention time of 23.5 minutes and a wavelength of 330 nm. The content of nodakenin was 3.11 ± 0.10 mg/g.

Experimental Example 1. Analysis of the effect of the mixed extract of the present invention and nodakenin on improving macrophage immunostimulatory activity

In order to evaluate the role of the mixed extract of the present invention and nodakenin in macrophage function, the cytotoxic effects of SH003 and nodakenin on RAW264.7 macrophages were first treated with various concentrations and evaluated by MTT assay. The results are shown in Figure 2a.

As shown in Figure 2 a, it was confirmed that 500 μg/ml of SH003 and 20 μg/ml of noda-kenin significantly reduced the cell viability of RAW264.7 macrophages.

Next, immune stimulation was performed by measuring the production levels of G-CSF (Granulocyte-Colony Stimulating Factor), IL-12, IL-2, IL-6, and TNF-α secreted by RAW264.7 macrophages. We wanted to investigate activity. Cytokine levels were measured using enzyme-linked immunosorbent assay. The results are shown in Figures 2b to 2f.

As shown in Figures 2b to 2f, SH003 was confirmed to increase the production levels of G-CSF, IL-12, IL-2, IL-6, and TNF-α in a concentration-dependent manner. Additionally, it was confirmed that nodakenin significantly increased the production levels of IL-12 and IL-2.

Next, we sought to analyze NO production in RAW264.7 macrophages cultured with SH003 or nodakenin. NO production was analyzed using a kit, and the mRNA and protein expression levels of iNOS were analyzed using RT-PCR and Western blot analysis.

The results are shown in Figure 3.

As shown in Figure 3a, SH003 was confirmed to increase NO levels in a concentration-dependent manner.

Additionally, as shown in Figure 3b and Figure 3c, the results confirmed that SH003 resulted in a significant increase in the mRNA and protein expression levels of iNOS, consistent with NO levels.

Subsequently, Western blotting and fluorescence staining were used to investigate whether SH003 and nodakenin induce NF-κB activation in RAW264.7 macrophages. The results are shown in Figure 4.

As shown in Figure 4 a, it was confirmed that SH003 or nodakenin increased the expression levels of NF-κB p65 in the nucleus and pIkBα in the cytoplasm, similar to the positive control (LPS). Additionally, as shown in Figure 4b, it was confirmed that SH003 or nodakenin induced nuclear translocation of NF-κB p65, consistent with nuclear NF-κB expression, similar to the positive control (LPS).

Experimental Example 2. Analysis of the effect of the mixed extract of the present invention and nodakenin on improving spleen cell immunostimulatory activity

We aimed to evaluate the immune-enhancing effects of SH003 and nodakenin in mouse primary spleen cells. The results are shown in Figure 5.

As shown in Figure 5, SH003 was confirmed to significantly increase the production levels of IFN-γ, IL-12, IL-2, and TNF-α in spleen cells. Additionally, it was confirmed that nodakenin significantly increased the production level of IL-12 in spleen cells.

Subsequently, we sought to further confirm whether SH003 and nodakenin improve the secretion level of NO in spleen cells. The results are shown in Figure 6.

As shown in Figure 6a, it was confirmed that SH003 improved NO levels similar to the positive control (LPS) in a concentration-dependent manner. In addition, as shown in Figure 6b and Figure 6c, SH003 was confirmed to cause a significant increase in the mRNA and protein expression levels of iNOS, similar to the positive control (LPS).

Measurement of lymphoproliferation is widely used to assess cellular function of the immune system (Nikbakht et al., 2019). Therefore, we sought to determine whether SH003 and nodakenin improve the proliferation of splenic lymphocytes by performing BrdU incorporation analysis using the BrdU cell proliferation assay kit (BioVision Inc., Milpitas, CA, USA). The results are shown in Figure 7.

As shown in Figure 7 (a), it was confirmed that SH003 or nodakenin significantly increased the incorporation of BrdU into proliferating cells. Additionally, as shown in Figure 7b, it was confirmed that SH003 or nodakenin increased the mRNA expression level of the endogenous nuclear protein Ki-67 as a marker of cell proliferation. As shown in Figure 7c, confocal immunofluorescence images confirmed Ki-67+ nuclei increased by SH003 or nodakenin treatment.

NK cells play an important role in immune defense against viral infections and tumors (Cook et al., 2014). Therefore, using the lactate dehydrogenase (LDH) cytotoxicity assay kit, we aimed to investigate whether SH003 and nodakenin enhance splenic NK cell activity by performing an LDH assay to evaluate cytotoxicity against NK cell-sensitive YAC-1 cells. did. The results are shown in Figure 8.

As shown in Figure 8, SH003 or nodakenin treatment significantly increased NK cell activity of splenocytes against YAC-1 cells (P < 0.05).

Experimental Example 3. Analysis of the effect of the mixed extract of the present invention and nodakenin in alleviating CP-induced immune suppression

As an antitumor drug, CP has immunosuppressive effects and has been frequently used to determine its immune-enhancing effects (Brodsky, 2010; Han et al., 2021a; Wang et al., 2018; Yang et al., 2019). Therefore, we sought to determine whether SH003 and nodakenin have an immunostimulatory effect under physiological conditions using a CP-induced immunosuppression mouse model.

The procedures used in animal research followed the guidelines approved by the Kyung Hee University Animal Care Committee (No. KHSASP-20-472) and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23. Revised, 1985). After 1 week of acclimatization, BALB/c mice (6-week-old male) were randomly assigned to 6 groups (n = 6/group). The six groups consisted of the blank group (Mice administered with PBS), CP control group (Sigma-Aldrich Co.), CP+SH003, CP+Nodakenin, CP+Red Ginseng (RG), and CP+G-CSF. The schematic diagram of administration to mice is shown in Figure 9A.

As shown in Figure 9a, 400 mg/kg of SH003, 10 mg/kg of nodakenin, and 400 mg/kg of red ginseng by oral administration or 1 μg/kg of G-CSF by intraperitoneal administration were administered for 7 consecutive days. RG and G-CSF were used as positive controls.

After the last administration, mice were weighed and then sacrificed by cervical dislocation. Blood and spleen were collected for further study. Relative spleen weight was calculated using the formula (spleen index (%) = (spleen weight (g) / body weight (g)) x 100). The results are shown in Table 1, Figure 9, and Figure 10 below.

Body weight change Spleen index Blank 3.29 ± 0.07 0.55 ± 0.02 CP control 0.41 ± 0.09*** 0.41 ± 0.01*** CP+SH003 2.28 ± 0.20### 0.69 ± 0.01### CP + nodakenin 1.51 ± 0.07### 0.69 ± 0.02### CP+RG 1.29 ± 0.02### 0.68 ± 0.01### CP+G-CSF 1.78 ± 0.11### 0.68 ± 0.01###

As shown in Table 1 and Figure 9b, the body weight change and spleen index of the CP control group were confirmed to be lower than those of the blank group, and the body weight change and spleen index of the SH003-treated CP mouse group and the nodakenin-treated CP mouse group were compared to the CP control group. It was confirmed that it was significantly higher.

In addition, as shown in Figure 9c to Figure 9f, the serum levels of IFN-γ, IL-12, IL-2, IL-6, and TNF-α were significantly decreased in the CP control group compared to the blank group. It was confirmed that there was a significant increase when treated with SH003 or nodakenin.

Meanwhile, as shown in Figure 10 (a) and Figure 10 (b), it was confirmed that SH003 or nodakenin treatment significantly increased the levels of IL-12 and IL-6 in the spleen, which were reduced by CP.

In addition, as shown in Figure 10c, the spleen cells in the blank group were dense, whereas the spleen cells in the CP control group were sparse and the spleen had necrotic areas, but the SH003 or nodakenin-treated CP mouse groups had necrotic areas in the spleen. It was confirmed that this was small and the spleen-spheric cells were dense.

In summary, it was confirmed that the mixed extract of Astragalus astragalus, Angelica root, and Chrysanthemum pollen of the present invention has a significant therapeutic effect on immunosuppression due to upregulation of immunostimulatory cytokines and NO levels through the NF-κB signaling pathway. Treatment with SH003 and nodakenin promoted splenic lymphocyte proliferation and splenic NK cell activity, and these effects were closely related to immune enhancement due to upregulation of immunostimulatory cytokines in the serum and spleen of CP-induced immunosuppressed mice. was confirmed. In other words, it was confirmed that the mixed extract of the present invention can have a therapeutic effect on immunosuppression.

From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (12)

A pharmaceutical composition for enhancing immunity containing mixed extracts of astragalus, angelica root, and perennial pollen as active ingredients.
The method of claim 1, wherein the mixed extract of Astragalus, Angelica root, and Astragalus root exhibits the ability to promote the production of granulocyte colony formation promoting factors, IL-12, IL-2, IL-6, TNF-α, or NO in macrophages. Pharmaceutical composition for enhancing immunity.
The method of claim 1, wherein the mixed extract of Astragalus, Angelica root, and Chrysanthemum pollen has the ability to promote proliferation, NK cell activity, and the ability to promote the production of IFN-γ, IL-12, IL-2, TNF-α, or NO in spleen cells. A pharmaceutical composition for enhancing immunity.
The method of claim 1, wherein the mixed extract is a pharmaceutical for enhancing immunity, which is obtained by extracting a mixture of Astragalus, Angelica root, and Chrysanthemum pollen with one or more solvents selected from the group consisting of water, alcohols with 1 to 4 carbon atoms, and mixed solvents thereof. Composition.
The pharmaceutical composition for enhancing immunity according to claim 4, wherein the alcohol having 1 to 4 carbon atoms is 20 to 40% (v/v) ethanol.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the mixed extract of Astragalus, Angelica root, and Astragalus root is mixed in a weight ratio of Astragalus: Angelica root: Angelica root: 0.5 to 5:1:1.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
The method of claim 1, wherein the composition is administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration. A pharmaceutical composition for enhancing immunity.
A method for enhancing immunity comprising administering the composition of any one of claims 1 to 8 to a subject other than a human.
A food composition for enhancing immunity containing mixed extracts of astragalus, angelica root, and wild pollen as active ingredients.
A pharmaceutical composition for preventing or treating immune diseases comprising a mixed extract of astragalus, angelica root, and wild pollen as an active ingredient.
The method of claim 11, wherein the immune disease includes cold, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergy, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, and red blood cell disease. A pharmaceutical composition for preventing or treating an immune disease selected from the group consisting of lupus and psoriasis.