Immune-enhancing composition containing mixed herbal extract as an active ingredient

Abstract

The present invention relates to a pharmaceutical composition for enhancing immunity, which contains a mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen as an active ingredient, and a method for enhancing immunity using said pharmaceutical composition.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for enhancing immunity, which contains a mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen as an active ingredient, and a method for enhancing immunity using said pharmaceutical composition. [Background technology]

[0002] Immune function regulation is a term that comprehensively encompasses the action of regulating and restoring normal immune function in the body to normal, or mitigating changes induced by environmental pollutants, side effects of medicines, disease, and aging. It is generally classified into immunosuppressive effects, which are regulatory effects that suppress undesirably increased immune responses, and immunoenhancing effects, which are regulatory effects that increase decreased immune responses.

[0003] Immunostimulation is an important therapeutic strategy for strengthening the body's defense mechanisms against various diseases, including cancer and inflammatory disorders. It can be achieved by increasing the activity of immune cells to stimulate the immune response. For example, macrophages play a key role in the immune response. Their primary role, phagocytosis, is to absorb microorganisms and other heat-generating particles, and to stimulate the immune response by secreting numerous cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-12 (IL-12), as well as 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 of enhancing the immune response. Since infections and diseases occur primarily when the immune system is weakened, various studies are being conducted to enhance immune responses through immune-enhancing substances when the body's immune system is weakened.

[0004] On the other hand, macrophages are known to be involved in various host responses, including intrinsic immunity as well as adaptive immunity, and are involved in host defense and homeostasis. During immune responses, they produce cytokines such as IL-1β (interleukin-1β), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α), and are known to play an important role in host defense in the early stages of infection (Journal of the Pharmaceutical Society of Japan 2013;57(6):394-399).

[0005] Under these circumstances, the present inventors have conducted extensive research to develop a formulation that is safe yet effective in enhancing immunity. As a result, they have confirmed that a mixed herbal extract of Astragalus Root, Angelica Root, and Angelica Root Pollen effectively enhances immunity, and have completed the present invention. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent 10-1245328 [Non-patent literature]

[0007] [Non-Patent Document 1] Wolf et al., 1994; Lee and Hong, 2011; Murray and Wynn, 2011 [Non-patent document 2] Journal of the Pharmaceutical Society 2013;57(6):394~399 [Non-patent document 3] Brodsky, 2010; Han et al., 2021a; Wang et al., 2018; Yang et al., 2019 Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have developed a pharmaceutical composition for enhancing immunity containing a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient, which can be used as an immune enhancer and an immune stimulant for various diseases caused or aggravated by immune deficiency, and a method for enhancing immunity using the pharmaceutical composition, thereby completing the present invention. [Means for solving the problem]

[0009] One object of the present invention is to provide a pharmaceutical composition for enhancing immunity, which contains a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient.

[0010] In any one specific example of the pharmaceutical composition for immune enhancement of the present invention, the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen may exhibit the ability to promote the production of granulocyte colony-stimulating factor, IL-12, IL-2, IL-6, TNF-α or NO in macrophages.

[0011] In any one specific example of the pharmaceutical composition for enhancing immunity of the present invention, the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen may exhibit the ability to promote proliferation, NK cell activity, and IFN-γ, IL-12, IL-2, TNF-α or NO production in spleen cells.

[0012] In any one specific example of the pharmaceutical composition for immune enhancement of the present invention, the mixed extract may be obtained by extracting a mixture of Astragalus Root, Angelica Root and Angelica Root pollen from one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms and mixed solvents thereof.

[0013] As a specific example of any one of the above-mentioned specific examples, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v / v) ethanol.

[0014] In any one specific example of the pharmaceutical composition for enhancing immunity of the present invention, the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen may be mixed in a weight ratio of Astragalus Root:Angelica Root:Angelica Root = 0.5 to 5:1:1.

[0015] In any one embodiment of the pharmaceutical composition for enhancing immunity of the present invention, the composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0016] In any one specific example of the pharmaceutical composition for immune enhancement of the present invention, the composition may be administered via intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration route.

[0017] Another object of the present invention is to provide a method for enhancing immunity, which comprises administering the composition to a non-human individual.

[0018] Another object of the present invention is to provide an immune-enhancing food composition containing a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating immune diseases, which comprises a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient.

[0020] In any one specific example of the pharmaceutical composition for preventing or treating an immune disease of the present invention, the immune disease may be selected from the group consisting of cold, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergy, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, and psoriasis. [Effects of the Invention]

[0021] The mixed extract of Astragalus chinensis, Angelica acutiloba and Angelica acutiloba of the present invention exhibits the effect of activating macrophages, spleen cells and NK cells, and is therefore useful as an immune enhancer and an immune stimulant for various diseases caused or aggravated by immune deficiency. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the results of HPLC analysis of nodakenin detected in the mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen of the present invention. [Figure 2] FIG. 1 shows the results of analyzing the effect of the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen of the present invention or nodakenin on improving macrophage immunostimulatory activity. [Figure 3] FIG. 1 shows the results of analyzing NO production in macrophages in response to the mixed extract of Astragalus membranaceus, Angelica acutiloba and Angelica acutiloba pollen of the present invention or nodakenin. [Figure 4] FIG. 1 shows the results of analyzing the NF-κB activation effect in macrophages of nodakenin, a mixed extract of Astragalus Root, Angelica Root, and Angelica Root pollen according to the present invention. [Figure 5] FIG. 1 shows the results of analyzing the effect of the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen of the present invention or nodakenin on improving the immunostimulatory activity of spleen cells. [Figure 6] FIG. 1 shows the results of analyzing NO production in spleen cells in response to the mixed extract of Astragalus Root, Angelica Root, and Angelica Root pollen of the present invention or nodakenin. [Figure 7] FIG. 1 shows the results of analyzing the effect of the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen of the present invention or nodakenin on promoting proliferation of splenic lymphocytes. [Figure 8] FIG. 1 shows the results of analyzing the effect of the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen of the present invention or nodakenin on promoting NK cell activity in spleen cells. [Figure 9] FIG. 1 shows the effect of the mixed extract of Astragalus membranaceus, Angelica acutiloba and Angelica acutiloba pollen of the present invention or nodakenin in alleviating CP-induced immunosuppression in a mouse model of CP-induced immunosuppression. [Figure 10] FIG. 1 shows the effect of the mixed extract of Astragalus membranaceus, Angelica acutiloba and Angelica acutiloba pollen of the present invention or nodakenin on alleviating CP-induced immunosuppression in the spleen of a mouse model of CP-induced immunosuppression. DETAILED DESCRIPTION OF THE INVENTION

[0023] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in the present invention also applies to each other description and embodiment. That is, any combination of various elements disclosed in the present application falls within the scope of the present invention. Furthermore, the following specific description is not considered to limit the category of the present application.

[0024] One aspect of the present invention is to provide a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient.

[0025] In the present invention, the term "Astragalus membranaceus" refers to the plant with the scientific name "Astragalus membranaceus"; "Angelica gigas" refers to the plant with the scientific name "Angelica gigas"; and "Trichosanthes kirilowii Maximowicz" refers to the plant with the scientific name "Trichosanthes kirilowii Maximowicz".

[0026] Astragalus membranaceus is a perennial plant of the dicotyledonous Rosales family, Fabaceae, and is found in Korea, Japan, Manchuria, northeastern China, and eastern Siberia. It is commonly cultivated as a medicinal herb, and in traditional Chinese medicine, it is harvested in the fall, the heads and roots removed, and dried in the sun. It is known for its medicinal properties when used whole, without peeling. It has stimulating, antiperspirant, diuretic, and antiswelling properties, and is prescribed for physical weakness, fatigue, and cold sweats.

[0027] Dong qui (Angelica gigas) is the dried root of the perennial herb of the Apiaceae family, known for its sweet and spicy taste and warming properties. One of the benefits of Dong qui is its blood-replenishing effect, which promotes blood flow in the coronary arteries and stimulates red blood cell production. It has also been reported that Dong qui extract containing decursin and / or decursinol anzelate can be used as an anticancer agent (Korean Patent Registered No. 10-1245328).

[0028] Trichosanthes kirilowii Maximowicz (Trichosanthes kirilowii Maximowicz) is the peeled root of the perennial trident or bitter gourd, which belongs to the Cucurbitaceae family. It has no odor, a bitter and sour taste, and is cold in nature. It is used to treat thirst, swelling, and pus when fluids are damaged by heat. It mainly reduces heat in the lungs and stomach, builds fluids, relieves thirst, and provides comfort to the body.

[0029] In the present invention, the ethanol extracts of Astragalus Root, Angelica Root and Angelica Root Pollen are used synonymously with "SH003" and can be used in combination.

[0030] In the present invention, the Astragalus Root, Dong Qi, or Angelica Root pollen may be purchased commercially, collected from nature, or cultivated, but is not limited thereto. The Astragalus Root, Dong Qi, or Angelica Root pollen extract may be extracted from natural, hybrid, or variant plants, or may be extracted from plant tissue culture.

[0031] The term "extract" as used herein includes the extract itself and all forms of extract that can be prepared using the extract, such as the extract obtained by extracting a mixture of Astragalus Root, Angelica Root, and Angelica Root Pollen, diluted or concentrated solutions of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, a fraction of the extract, or a mixture thereof.

[0032] In one specific example of the pharmaceutical composition for immune enhancement of the present invention, the mixed extract may be extracted from a mixture of Astragalus Root, Angelica Root and Angelica Root pollen with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms and mixed solvents thereof, specifically, ethanol extract, but is not limited thereto.

[0033] As a specific example of any one of the above-mentioned specific examples, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v / v) ethanol, specifically 30% (v / v) ethanol, but is not limited thereto.

[0034] In the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, the method for extracting the mixture is not particularly limited and can be any method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., which can be performed alone or in combination of two or more methods.

[0035] In addition, the extract may be prepared in the form of a dry powder after extraction and used, but is not limited thereto.

[0036] In one specific example of the pharmaceutical composition for immune enhancement of the present invention, the mixed extract of Astragalus chinensis, Angelica acutiloba and Angelica acutiloba may be mixed in a weight ratio of Astragalus chinensis:Angelica acutiloba:Angelica acutiloba = 0.5 to 5:1:1, specifically, a weight ratio of Astragalus chinensis:Angelica acutiloba:Angelica acutiloba = 1 to 3:1:1, more specifically, a weight ratio of Astragalus chinensis:Angelica acutiloba:Angelica acutiloba = 1:1:1, but is not limited thereto.

[0037] The term "immune enhancement" as used herein refers to enhancing the body's defense against antigens, specifically, enhancing cellular and humoral immunity against antigens. The mechanism of immune enhancement is not limited, but may include, for example, enhancing the activity of antigen-presenting cells such as macrophages or enhancing specific activity of lymphocytes.

[0038] In any one specific example of the pharmaceutical composition for enhancing immunity of the present invention, the mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen may exhibit the ability to promote the production of granulocyte colony-stimulating factor, IL-12, IL-2, IL-6, TNF-α or NO in macrophages.

[0039] In any one specific example of the pharmaceutical composition for enhancing immunity of the present invention, the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen may exhibit the ability to promote proliferation of spleen cells, the ability to promote NK cell activity, and the ability to promote the production of IFN-γ, IL-12, IL-2, TNF-α or NO in spleen cells.

[0040] The immunopotentiating pharmaceutical composition of the present invention can improve immunosuppression through activation of macrophages, splenocytes and NK cells.

[0041] The active ingredient of the mixed extract of Astragalus Root, Dong Qi, and Angelica Root Pollen may be, but is not limited to, nodakenin. Nodakenin can be isolated from natural sources such as the mixed extract of Astragalus Root, Dong Qi, and Angelica Root Pollen, but is not limited to this, and may be chemically synthesized by methods known in the art or may be a commercially available substance.

[0042] The pharmaceutical composition of the present invention may contain the extract in an amount of 0.001 to 80, specifically 0.001 to 70, more specifically 0.001 to 60 wt % based on the total weight of the composition, but is not limited thereto.

[0043] In any one specific example of the pharmaceutical composition for immune enhancement of the present invention, the composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent, but is not limited thereto.

[0044] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier.

[0045] The term "pharmaceutically acceptable" as used herein means that the composition exhibits the property of being non-toxic to cells or humans exposed to the composition.

[0046] Specifically, the pharmaceutical compositions can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injection solutions according to conventional methods. In the present invention, carriers, excipients, and diluents contained in the pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and are formulated by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid dosage forms include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Parenteral dosage forms include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0047] In any one specific example of the pharmaceutical composition for immune enhancement of the present invention, the composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration, but is not limited to these.

[0048] Another aspect of the present invention provides a method for enhancing immunity, comprising administering the composition to a non-human individual.

[0049] The term "administration" as used herein means introducing a given substance into an individual by an appropriate method.

[0050] The term "individual" as used herein refers to any animal, including humans, whose immune system is desired to be enhanced, such as mice, rats, livestock, etc. Specific examples include, but are not limited to, mammals, including humans.

[0051] Specifically, the method for enhancing immunity of the present invention may include administering to an individual other than a human a pharmaceutically effective amount of a pharmaceutical composition containing a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen as an active ingredient.

[0052] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and which does not cause side effects. The effective dose level can be easily determined by one skilled in the art depending on factors including the patient's sex, age, weight, health condition, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route, and excretion rate, treatment period, concomitant or concomitant drugs, and other factors well known in the medical field.

[0053] The pharmaceutical composition may be administered alone or in combination with other immune-enhancing preparations, and may be administered sequentially or simultaneously with conventional immune-enhancing preparations. It may also be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that will provide the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0054] For example, the composition of the present invention may be administered at a dose of 0.0001 to 100 mg / kg of body weight, more specifically 0.001 to 100 mg / kg of body weight, based on the solid content, per day. The recommended dose may be administered once a day or in divided doses.

[0055] In addition, the administration route and mode of administration of the composition are not particularly limited and may be any administration route and mode as long as the composition containing the composition can be delivered to the desired site. Specifically, the composition may be administered through various routes, such as oral or parenteral, including but not limited to oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, or inhalation. The dosage may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the administration route, and the administration time, but may be appropriately selected by those skilled in the art.

[0056] Another aspect of the present invention provides an immune-enhancing food composition comprising a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient.

[0057] The term "food" as used herein includes all foods in the ordinary sense, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, vitamin complexes, functional health foods, and health foods.

[0058] Since the food composition of the present invention is derived from a mixed extract of Astragalus Root, Angelica Root, and Angelica Root pollen, which can be ingested on a daily basis, it is expected to have a high immune-enhancing effect and is very useful for the purpose of promoting health.

[0059] The term "functional food" is similar to "food for special health use (FoSHU)" and refers to a food with high medical and therapeutic effects that is processed to efficiently exert bioregulatory functions in addition to providing nutrients. Here, "functionality" refers to regulating nutrients to the structure and function of the human body and achieving beneficial health effects such as physiological effects. The food of the present invention can be produced by methods commonly used in the art, and can be produced by adding raw materials and ingredients commonly added in the art. Furthermore, the food can be produced in any dosage form recognized as a food without any restrictions.

[0060] The food composition of the present invention may be manufactured in various dosage forms. Unlike over-the-counter medicines, it has the advantage of being made from natural ingredients and is free of side effects that can occur when taking medicines for a long period of time. Furthermore, because it is highly portable, the food of the present invention can be taken as a supplement to enhance immunity.

[0061] The term "health food" refers to food that has more active health maintenance and promotion effects than general foods, and "health supplement food" refers to food intended for health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement" are used interchangeably.

[0062] Specifically, the health functional foods are foods in which the compounds of the present invention are added to food ingredients such as beverages, teas, spices, gums, and confectioneries, or are prepared as capsules, powders, suspensions, etc., and when ingested, they bring about specific health benefits. However, unlike over-the-counter medicines, they have the advantage of being made from food ingredients and therefore free from side effects that can occur when taking medicines for a long period of time.

[0063] The food composition may further contain a physiologically acceptable carrier, but the type of carrier is not particularly limited, and any carrier commonly used in the art can be used.

[0064] The food composition may also contain additional ingredients commonly used in food compositions to improve smell, taste, visual appearance, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr) may also be included; and amino acids such as lysine, tryptophan, cysteine, and valine may also be included.

[0065] The food compositions may also contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium D-bitartrate, etc.), fortifiers, emulsifiers, thickeners (thickeners), coating agents, gum bases, foam inhibitors, solvents, and improvers. The additives may be selected depending on the type of food and used in appropriate amounts.

[0066] An example of the food composition of the present invention may be a health drink composition. In this case, various flavorings or natural carbohydrates may be added as additional ingredients, as in conventional beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may generally be 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.

[0067] In addition to the above, the health drink composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectinic acid, pectinic acid salts, alginic acid, alginic acid salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. Fruit pulp may also be included for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is typically selected from 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.

[0068] Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating immune diseases, comprising a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen as an active ingredient.

[0069] The term "prevention" in the present invention refers to any action of suppressing or delaying immune diseases by administering the immunopotentiating pharmaceutical composition containing the mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an active ingredient.

[0070] The term "treatment" as used herein means any action in which the symptoms of individuals suspected of or affected by an immune disease are improved or beneficially altered by administering the pharmaceutical composition.

[0071] The term "immune disease" as used herein includes various inflammatory diseases, allergic diseases, cancers, and the like caused by abnormalities in the immune system of the living body, and may include, but is not limited to, colds, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergies, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, and psoriasis.

[0072] The composition of the present invention may contain one or more known active ingredients having immune-enhancing or immune disease preventive and therapeutic effects, together with the mixed extract of Astragalus Root, Angelica Root, and Angelica Root pollen.

[0073] The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for immune enhancement or prevention and treatment of immune disorders.

[0074] Example Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding the present invention. However, the examples according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0075] Example 1. Preparation of fractions of mixed herbal ethanol extract (SH003) and nodakenin Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes kirilowii Maximowicz (Tk) were mixed in a 1:1:1 weight ratio (w / w), placed in an extractor, and extracted with 30% (v / v) ethanol at 100°C for 3 hours. The extract was filtered, and the filtrate was concentrated under reduced pressure and dried to obtain a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz. The dried extract was dissolved in 30% ethanol and stored at -80°C until use. When used, it was redissolved in 30% ethanol to a concentration of 100 mg / ml and further diluted with culture medium.

[0076] Nodakenin (purity ≥97%, Ensol Biosciences Inc., Daejeon, Korea) was prepared according to a previous study (Li et al., 2020). Before use, nodakenin was dissolved in dimethyl sulfoxide (DMSO) and further diluted in culture medium (DMSO <0.2%).

[0077] Example 2. Analysis of nodakenin To standardize the SH003 extract for the presence of nodakenin, the nodakenin in SH003 was analyzed using an HPLC system (Shimadzu 20A, Shimadzu Co., Kyoto, Japan). The 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.

[0078] As shown in Figure 1, nodakenin was detected at a wavelength of 330 nm with a retention time of 23.5 minutes. The nodakenin content was 3.11±0.10 mg / g.

[0079] Experimental Example 1. Analysis of the effect of the mixed extract of the present invention and nodakenin on improving macrophage immunostimulatory activity To evaluate the role of the inventive mixed extract and nodakenin on macrophage function, the cytotoxic effects of SH003 and nodakenin on RAW264.7 macrophages were evaluated by MTT assay after treatment with various concentrations. The results are shown in Figure 2a.

[0080] As shown in Figure 2a, it was confirmed that SH003 at 500 μg / ml and noda-kenin at 20 μg / ml significantly reduced the cell viability of RAW264.7 macrophages.

[0081] We then investigated the immunostimulatory activity of the RAW264.7 macrophages by measuring the production levels of Granulocyte-Colony Stimulating Factor (G-CSF), IL-12, IL-2, IL-6, and TNF-α. Cytokine levels were measured using an enzyme-linked immunosorbent assay. The results are shown in Figure 2b to Figure 2f.

[0082] As shown in Figure 2b to Figure 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. In addition, nodakenin was confirmed to significantly increase the production levels of IL-12 and IL-2.

[0083] We then analyzed NO production in RAW264.7 macrophages cultured with SH003 or nodakenin. NO production was analyzed using a kit, and iNOS mRNA and protein expression levels were analyzed using RT-PCR and Western blot analysis.

[0084] The results are shown in Figure 3.

[0085] As shown in FIG. 3a, SH003 was confirmed to increase NO levels in a concentration-dependent manner.

[0086] Furthermore, as shown in Figure 3b and Figure 3c, the results confirmed that SH003 resulted in a significant increase in iNOS mRNA and protein expression levels, which coincided with NO levels.

[0087] Next, we investigated whether SH003 and nodakenin induce NF-κB activation in RAW264.7 macrophages using Western blotting and fluorescent staining, and the results are shown in Figure 4.

[0088] As shown in Figure 4(a), SH003 or nodakenin, like the positive control (LPS), increased the expression levels of NF-κB p65 in the nucleus and pIkBα in the cytoplasm. Furthermore, as shown in Figure 4(b), SH003 or nodakenin, like the positive control (LPS), induced a nuclear potential for NF-κB p65, which coincided with nuclear NF-κB expression.

[0089] Experimental Example 2. Analysis of the effect of the mixed extract of the present invention and nodakenin on improving the immunostimulatory activity of spleen cells We evaluated the immunopotentiating effects of SH003 and nodakenin on primary mouse splenocytes, and the results are shown in Figure 5.

[0090] As shown in Figure 5, SH003 significantly increased the production levels of IFN-γ, IL-12, IL-2, and TNF-α in splenocytes, and nodakenin significantly increased the production level of IL-12 in splenocytes.

[0091] Next, we further investigated whether SH003 and nodakenin could increase the secretion level of NO in splenocytes. The results are shown in Figure 6.

[0092] As shown in Figure 6(a), SH003 increased NO levels in a concentration-dependent manner, similar to the positive control (LPS). Also, as shown in Figure 6(b) and Figure 6(c), SH003 significantly increased iNOS mRNA and protein expression levels, similar to the positive control (LPS).

[0093] Measurement of lymphocyte proliferation is widely used to evaluate the cellular function of the immune system (Nikbakht et al., 2019). Therefore, we performed BrdU integration analysis using a BrdU Cell Proliferation Assay Kit (BioVision Inc., Milpitas, CA, USA) to determine whether SH003 and nodakenin improved splenic lymphocyte proliferation. The results are shown in Figure 7.

[0094] As shown in Figure 7(a), SH003 or nodakenin significantly increased BrdU incorporation into proliferating cells. Furthermore, as shown in Figure 7(b), SH003 or nodakenin increased the mRNA expression level of the endogenous nuclear protein Ki-67, a marker of cell proliferation. As shown in Figure 7(c), confocal immunofluorescence images confirmed increased Ki-67+ nuclei following treatment with SH003 or nodakenin.

[0095] NK cells play an important role in immune defense against viral infections and tumors (Cook et al., 2014). Therefore, we performed a lactate dehydrogenase (LDH) cytotoxicity assay kit to evaluate cytotoxicity against NK cell-sensitive YAC-1 cells and investigated whether SH003 and nodakenin enhanced splenic NK cell activity. The results are shown in Figure 8.

[0096] As shown in FIG. 8, treatment with SH003 or nodakenin significantly increased the NK cell activity of splenocytes against YAC-1 cells (P<0.05).

[0097] Experimental Example 3. Analysis of the effect of the mixed extract of the present invention and nodakenin on alleviating CP-induced immunosuppression 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 decided to use a CP-induced immunosuppressed rat model to determine whether SH003 and nodakenin have immune-stimulating effects under physiological conditions.

[0098] The procedures used in animal studies were in accordance with 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 one week of acclimation, BALB / c mice (6-week-old male) were randomly assigned to six groups (n = 6 per group). The six groups consisted of a blank group (PBS-treated mice), a CP control group (Sigma-Aldrich Co.), CP + SH003, CP + nodakenin, CP + red ginseng (RG), and CP + G-CSF. A schematic diagram of the mouse administration is shown in Figure 9(a).

[0099] As shown in Figure 9(a), 400 mg / kg of SH003, 10 mg / kg of nodakenin, 400 mg / kg of red ginseng, or 1 μg / kg of G-CSF were administered intraperitoneally for 7 consecutive days. RG and G-CSF were used as positive controls.

[0100] After the final administration, the mice were weighed and then sacrificed by cervical dislocation. Blood and spleens 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 below and Figures 9 and 10.

[0101] [Table 1]

[0102] As shown in Table 1 and Figure 9b, the weight change and spleen index of the CP control group were lower than those of the blank group, while the weight change and spleen index of the SH003-treated CP mouse group and the nodakenin-treated CP mouse group were significantly higher than those of the CP control group.

[0103] Furthermore, as shown in Figure 9c to Figure 9f, it was confirmed that 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, whereas they were significantly increased upon treatment with SH003 or nodakenin.

[0104] On the other hand, as shown in Figure 10a and Figure 10b, it was confirmed that treatment with SH003 or nodakenin significantly increased the levels of IL-12 and IL-6 in the spleen, which had been decreased by CP.

[0105] Furthermore, 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, whereas the SH003 or nodakenin-treated CP mouse groups had fewer necrotic areas in the spleen and dense spleenocytes.

[0106] In summary, the present invention's mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen has significant therapeutic effects on immunosuppression through upregulation of immunostimulatory cytokines and NO levels via the NF-κB signaling pathway. Treatment with SH003 and nodakenin promoted splenic lymphocyte proliferation and splenic NK cell activity, and these effects were closely associated with immune enhancement through upregulation of immunostimulatory cytokines in the serum and spleen of CP-induced immunosuppressed mice. This confirms that the present invention's mixed extract may have therapeutic effects on immunosuppression.

[0107] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including within the meaning and scope of the claims below, and any modifications or alterations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A pharmaceutical composition for enhancing immunity, comprising a mixed extract of Astragalus chinensis, Angelica acutiloba and Angelica acutiloba pollen as an active ingredient.

2. 2. The pharmaceutical composition for enhancing immunity according to claim 1, wherein the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen exhibits the ability to promote the production of granulocyte colony-stimulating factor, IL-12, IL-2, IL-6, TNF-α, or NO in macrophages.

3. 2. The pharmaceutical composition for enhancing immunity according to claim 1, wherein the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen exhibits the ability to promote proliferation of spleen cells, the ability to promote NK cell activity, and the ability to promote the production of IFN-γ, IL-12, IL-2, TNF-α or NO in spleen cells.

4. 2. The pharmaceutical composition for immune enhancement according to claim 1, wherein the mixed extract is obtained by extracting a mixture of Astragalus Root, Angelica Root, and Angelica Root Pollen from one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

5. 5. 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.

6. 2. The pharmaceutical composition for enhancing immunity according to claim 1, wherein the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen is mixed in a weight ratio of Astragalus Root:Angelica Root:Angelica Root Pollen = 0.5 to 5:1:

1.

7. The pharmaceutical composition for enhancing immunity according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.

8. The pharmaceutical composition for immune enhancement according to claim 1, wherein the composition is administered by intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration.

9. A method for enhancing immunity, comprising administering the composition according to any one of claims 1 to 8 to an individual other than a human.

10. An immune-enhancing food composition comprising a mixed extract of Astragalus chinensis, Angelica acutiloba and Angelica acutiloba pollen as an active ingredient.

11. A pharmaceutical composition for preventing or treating immune diseases, comprising a mixed extract of Astragalus chinensis, Angelica acutiloba and Angelica acutiloba as an active ingredient.

12. 12. The pharmaceutical composition for preventing or treating an immune disease according to claim 11, wherein the immune disease is selected from the group consisting of colds, asthma, pneumonia, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, allergies, rheumatoid arthritis, Alzheimer's disease, autoimmune thyroid disease, inflammatory bowel disease, aplastic anemia, lupus erythematosus, and psoriasis.

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