Immunity-enhancing composition containing kyoho grapevine fruiting shoot extract as active ingredient

Abstract

The present invention relates to an immunity-enhancing composition containing a Kyoho grapevine fruiting shoot extract as an active ingredient. The Kyoho grapevine fruiting shoot extract according to the present invention exhibits an excellent effect in terms of increasing the nitric oxide (NO) and interferon-β (IFN-β) secretion of macrophages, and can thus be effectively used as a health functional food for enhancing immunity, an animal feed additive for enhancing immunity, or a pharmaceutical composition for preventing and treating diseases caused by immune deficiency.

Description

An immune-enhancing composition containing an extract of the fruit of Kyoho grapevine as an effective ingredient

[0001] The present invention relates to an immune-enhancing composition containing an extract of the fruit stem of Kyoho grapes as an effective ingredient.

[0002]

[0003] This work was supported by the National Research Foundation of Korea (NRF) under the Individual Basic Research Program of the Ministry of Science and ICT (Project Number: 2021R1A2C2094436).

[0004] Immunity is a series of self-protection mechanisms that the human body uses to prevent substances other than its own components from disrupting homeostasis or threatening the body. It is broadly divided into innate immunity (innate immunosurveillance) and adaptive immunity (adaptive immunosurveillance).

[0005] Innate immunity, also known as natural immunity, is composed of white blood cells including macrophages, natural killer cells, various cytokines such as TNF-α, mucous tissue, and complement present in the blood. It plays a primary defensive role by inducing phagocytosis against infectious pathogens such as microorganisms and viral invasions, as well as aged normal cells and cancer cells before the development of acquired immunity. Adaptive immunity, which plays a role in reinforcing innate immunity, is formed when T cells recognize antigens presented by antigen-presenting cells and engulf them. It consists of cellular immunity involving T cells and humoral immune response by antibodies produced by B cells. In this way, the human body is designed to maintain homeostasis by appropriately fighting against the invasion or infection of foreign substances such as external antigens through innate and adaptive immunity.

[0006] The spleen is a crucial immune organ that produces various immune cells, including lymphocytes, macrophages, and natural killer cells. It responds to blood-borne antigens and also removes aged and damaged cells. The thymus is a butterfly-shaped immune organ involved in the production and differentiation of lymphocytes.

[0007] In innate immunity, macrophages located in the spleen play the most important role. Macrophages are the first to recognize the invasion of foreign substances and participate in humoral and cellular immunity. When activated, they not only increase proliferation and diffusion ability and enhance phagocytic ability, but also secrete cytokines such as NO (nitric oxide), TNF-α, IL-1β, and IL-6, which inhibit the proliferation of infectious pathogens such as various microorganisms and viruses and are known to exhibit toxicity against cancer cells.

[0008] Meanwhile, a weakened immune system gradually weakens the body's inherent natural healing capabilities, leading to various diseases and symptoms. To address this, a fundamental approach to boosting immunity is essential. However, because immunity varies from person to person, a long-term approach is necessary. Various treatments and supplements aimed at boosting immunity have been developed and marketed. However, due to the side effects associated with long-term use, the use of relatively safer supplements, particularly natural supplements, is considered preferable to therapeutics. Active research is underway in this area.

[0009] Meanwhile, Kyoho is a grape variety that has been improved to have large, soft, and juicy grapes. It is a hybrid of Vitis vinifera, a European grape, and Vitis labrusca, a Native American grape. The variety was completely improved in Japan in 1942, and through this improvement, the sugar content of the juice is measured to be three to six times higher than that of regular grapes. The harvest season is from late August to mid-September, and the roots do not easily rot even in moist places. The Kyoho variety distributed in Korea is considered the standard variety with an average sugar content of about four times higher, distributed through the Korea Grape Association.

[0010] Meanwhile, as for technologies related to enhancing immunity, Korean Patent No. 2119178 discloses an 'immunity enhancing composition containing fermented extract of Angelica gigas Nakai as an active ingredient', and Korean Patent No. 2168310 discloses an 'immunity enhancing health functional food composition containing fermented extract of Aronia japonica', but the 'immunity enhancing composition containing extract of the fruit stem of Kyoho grape vine as an active ingredient' of the present invention has not been disclosed yet.

[0011] The present invention was derived from the above-mentioned needs, and the present invention provides an immune-enhancing composition containing an extract of the fruit stem of a Kyoho grape as an active ingredient, and confirmed that the extract of the fruit stem of a Kyoho grape of the present invention increases the secretion of NO (Nitric Oxide) and IFN-β (Interferon β) in macrophages, and confirmed that it increases the weight of spleen and thymus and increases the secretion of IFN-γ (Interferon-γ) and IL-12 (Interleukin-12) in blood in an immunosuppressed animal model induced by administration of cyclophosphamide, thereby completing the present invention.

[0012] In order to solve the above problem, the present invention provides a health functional food composition for enhancing immunity containing an extract of the fruit stem of Kyoho grape vine as an effective ingredient.

[0013] In addition, the present invention provides an immune-boosting feed additive containing an extract of the fruiting body of Kyoho grapes as an effective ingredient.

[0014] In addition, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by a decrease in immunity, containing an extract of the fruit of a Kyoho grape vine as an effective ingredient.

[0015] The present invention relates to an immune-enhancing composition containing an extract of the fruit stalk of a grapevine as an active ingredient, wherein the extract of the fruit stalk of a grapevine of the present invention has an excellent effect of increasing the amount of NO (Nitric Oxide) and IFN-β (Interferon β) secreted by macrophages compared to a control group that was not treated with anything and a group treated with the same concentration of a grapevine fruit stalk extract, and has an excellent effect of increasing the weight of the spleen and thymus and increasing the amount of IFN-γ (Interferon-γ) and IL-12 (Interleukin-12) secreted in the blood in an immunosuppressed animal model induced by administration of cyclophosphamide.

[0016] Figure 1 shows the results of confirming the NO (A) and IFN-β (B) secretion amounts of macrophages by treatment with the extract of the grapevine fruiting body or the extract of the grapevine fruiting body. LPS (Lipopolysaccharide) is an immune response inducer. *, *** indicate that the NO or IFN-β secretion amounts of the LPS-only treatment group, the grapevine fruiting body extract or the grapevine fruiting body extract administration group were statistically significantly increased compared to the LPS-free group. * is p<0.05, and *** is p<0.001.

[0017] Figure 2 shows the results of measuring the spleen weight (A) and thymus weight (B) in an immunocompromised animal model treated with the extract of the stem of the grapevine. Cyclophosphamide is an immunocompromised inducer. Normal is a normal group that was not administered anything, control is an immunocompromised-induced group, Gwangbong Yeolsu 50 is an experimental group that administered 50 mg / kg of the water extract of the stem of the grapevine to an immunocompromised animal model, Gwangbong Yeolsu 100 is an experimental group that administered 100 mg / kg of the water extract of the stem of the grapevine to an immunocompromised animal model, Gwangbong Ethanol 50 is an experimental group that administered 50 mg / kg of the ethanol extract of the stem of the grapevine to an immunocompromised animal model, and Gwangbong Ethanol 100 is an experimental group that administered 100 mg / kg of the water extract of the stem of the grapevine to an immunocompromised animal model. #, ## indicate that the weight of the spleen or thymus in the immunosuppressed group was statistically significantly reduced compared to the normal group, and # indicates p<0.05, ## indicates p<0.01, and * indicates that the weight of the spleen or thymus in the group administered with the extract of the Kyoho grapevine was statistically significantly increased compared to the immunosuppressed group, and p<0.05.

[0018] Figure 3 shows the results of confirming the amount of blood IFN-γ secretion (A) and IL-12 secretion (B) in an immunosuppressed animal model treated with the extract of the fruit stalks of Kyoho grapevine. Cyclophosphamide is an immunosuppressant inducer. Normal is a normal group that was not administered anything, control is an immunosuppressed-induced group, Kyoho Hot Water 50 is an experimental group that administered 50 mg / kg of the water extract of the fruit stalks of Kyoho grapevine to an immunosuppressed animal model, Kyoho Hot Water 100 is an experimental group that administered 100 mg / kg of the water extract of the fruit stalks of Kyoho grapevine to an immunosuppressed animal model, Kyoho Ethanol 50 is an experimental group that administered 50 mg / kg of the ethanol extract of the fruit stalks of Kyoho grapevine to an immunosuppressed animal model, and Kyoho Ethanol 100 is an experimental group that administered 100 mg / kg of the water extract of the fruit stalks of Kyoho grapevine to an immunosuppressed animal model. ### indicates that the IFN-γ or IL-12 secretion amount in the immunosuppressed group was statistically significantly decreased compared to the normal group, p<0.001, and *, *** indicate that the IFN-γ or IL-12 secretion amount in the group administered with the Kyoho grapevine fruit extract was statistically significantly increased compared to the immunosuppressed group, * indicates p<0.05, and *** indicates p<0.001.

[0019] In order to achieve the purpose of the present invention, the present invention provides a health functional food composition for enhancing immunity containing an extract of the fruit stem of Kyoho grape vine as an effective ingredient.

[0020] The bearing mother branch of the present invention is a part cut through pruning, and is the part from which the bearing mother branch, which is a branch bearing fruit, grows.

[0021] In the health functional food composition for enhancing immunity of the present invention, the extraction solvent of the fruit stem extract of the Kyoho grape vine may be water, a lower alcohol of C1 to C4, or a mixture thereof, but is not limited thereto.

[0022] The above composition is preferably prepared in any one of the following forms: powder, granules, pills, tablets, capsules, candies, syrups, and beverages, but is not limited thereto.

[0023] When the health functional food composition of the present invention is used as a food additive, the extract of the fruit stem of the Kyoho grape vine can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. There is no particular limitation on the type of the food. Examples of foods to which the above extract can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0024] When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotensin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 g of the composition of the present invention. The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in mixtures. The proportion of these additives is not particularly important, but the composition of the present invention is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

[0025] In addition, the present invention provides an immune-boosting feed additive containing an extract of the fruiting body of Kyoho grapes as an effective ingredient.

[0026] The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term "feed" in the present invention may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are intended for or suitable for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0027] In addition, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by a decrease in immunity, containing an extract of the fruit of a Kyoho grape vine as an effective ingredient.

[0028] The diseases caused by the above-mentioned weakened immunity are preferably at least one selected from among cold, asthma, sinusitis, pneumonia, sepsis, osteomyelitis, meningitis, herpes simplex, shingles, stomatitis, and psoriasis, but are not limited thereto.

[0029] The composition of the present invention may be prepared in any one dosage form selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto. The composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-described active ingredient, and may be in various oral or parenteral dosage forms. When formulated, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, and pills, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, and aerosols. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. For parenteral administration, it is preferable to select a method such as topical application to the skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection. The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount can be determined according to the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art. The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity, and the daily dosage is 0.01 to 2,000 mg / kg based on the amount of the complex herbal medicine extract, preferably 50 to 1,000 mg / kg, and more preferably 300 to 500 mg / kg, and can be administered 1 to 6 times a day. 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 modifiers.

[0030]

[0031] Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0032]

[0033] Manufacturing Example 1. Manufacturing of extract of Kyoho grape vine fruit

[0034] The present invention was obtained by pruning (pruning) the cut mother branches (bearing mother branches, the part from which the fruit-bearing branches grow) after harvesting Kyoho grapes in vineyards in Daegu and Gyeongbuk, Korea (2021). 10 times the amount of 70% (v / v) ethanol was added to 10 kg of the obtained mother branches of the Kyoho grape vine in a cut state, and the mixture was extracted twice by reflux cooling in a heating mantle at 75℃ for 7 days, and then the filtrate was filtered under reduced pressure through a Buchner funnel lined with filter paper, and the filtrate was concentrated under reduced pressure to obtain an ethanol extract of the mother branches of the Kyoho grape vine.

[0035] In addition, 2 L of distilled water was added to 750 g of the stem of the cut Kyoho grape vine, extracted in a 100°C water heater for 2 hours, and then the filtrate was filtered under reduced pressure through a Buchner funnel with a filter paper placed on it, and dried to obtain a hot water extract of the stem of the Kyoho grape vine.

[0036]

[0037] Example 1. Measurement of macrophage immune activity

[0038] RAW 264.7 cells were seeded in 6-well plates and cultured at 1 × 10 in DMEM (Dulbecco Modified Eagle Medium), 37°C, 5% CO2 conditions. 5 The cells were cultured until the cell / well concentration reached 100 μg / ml. Afterwards, the Kyoho grapevine extract of Manufacturing Example 1 was treated at concentrations of 1, 10, or 100 μg / ml, respectively, and cultured for 24 hours. An immune response was induced by treatment with 0.1 μg / ml of LPS (Lipopolysaccharide) and used as a positive control. After culturing, the supernatant of the culture was obtained, and the secretion amounts of NO (Nitric Oxide) and IFN-β (Interferon-β) were measured using an ELISA kit.

[0039] As a result, as disclosed in Fig. 1, the NO and IFN-β secretion amounts of the group treated with the resultant extract of the Kyoho grapevine of the present invention were significantly increased compared to the control group that was not treated with anything and the group treated with the resultant extract of the cornelian cherry grapevine at the same concentration, and were confirmed to be significantly increased in the hot water extract compared to the 70% ethanol extract.

[0040]

[0041] Example 2. Analysis of the effect of improving immune function in an immunocompromised animal model.

[0042] Five-week-old BALB / c mice (Youngnam Bio) were acclimatized for a week, and then animal experiments were conducted in compliance with the animal experiment ethics regulations of the Korea Institute of Oriental Medicine (Approval No. 23-108). The experiments were conducted with five mice per experimental group according to Table 1 below, and 100 mg / kg of cyclophosphamide was administered intraperitoneally to induce immunosuppression. Three hours after cyclophosphamide administration, the Kyoho grapevine stem extract of Preparation Example 1 was orally administered at a concentration of 50 or 100 mg / kg, respectively. The group that was not treated with anything was used as the normal group, and the group that was administered cyclophosphamide alone was used as the immunosuppression-induced group (control).

[0043] Animal experiment category: Induction of immunosuppression. Treatment with Kyoho grapevine extract. Normal--control. Cyclophosphamide 100mg / kg. Kyoho hot water 50mg / kg. Hot water extract 50mg / kg. Kyoho hot water 100mg / kg. Hot water extract 100mg / kg. Kyoho ethanol 50mg. Ethanol extract 50mg / kg. Kyoho ethanol 100mg. Ethanol extract 100mg / kg.

[0044]

[0045] 2-1. Measurement of spleen and thymus weight

[0046] After sacrificing the mice, the spleen and thymus were removed and weighed, and the relative weight compared to the body weight before sacrifice was calculated.

[0047] As a result, as disclosed in Fig. 2, it was confirmed that the weight of the spleen and thymus of the immunosuppressed group was significantly reduced compared to the normal group, and it was confirmed that the weight of the spleen and thymus of the group treated with the resultant extract of the Kyoho grape of the present invention was increased compared to the immunosuppressed group.

[0048]

[0049] 2-2. Measurement of blood IFN-γ (Interferon-γ) and IL-12 (Interleukin-12) secretion levels

[0050] Blood was collected from the iliac vein, centrifuged at 3,000 rpm for 10 minutes, and serum was recovered. The secretion levels of IFN-γ and IL-12 were measured using an ELISA kit.

[0051] As a result, as disclosed in Fig. 3, it was confirmed that the secretion amounts of IFN-γ and IL-12 in the immunosuppressed group were significantly reduced compared to the normal group, and it was confirmed that the secretion amounts of IFN-γ and IL-12 in the group treated with the extract of the resultant grape of the present invention were significantly increased compared to the immunosuppressed group.

Claims

1. A health functional food composition for enhancing immunity containing an extract of the fruit of Kyoho grape vine as an effective ingredient.

2. A health functional food composition for enhancing immunity, characterized in that the extraction solvent of the resultant extract of the Kyoho grapevine in paragraph 1 is water, a lower alcohol of C1 to C4, or a mixture thereof.

3. A health functional food composition for enhancing immunity, characterized in that the composition in claim 1 is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

4. Feed additive for enhancing immunity containing extract of Kyoho grape vine fruit as an effective ingredient.

5. A pharmaceutical composition for preventing or treating diseases caused by decreased immunity, containing an extract of the fruit of Kyoho grape vine as an effective ingredient.

6. A pharmaceutical composition for preventing or treating a disease caused by a weakened immune system, characterized in that in paragraph 5, the disease caused by a weakened immune system is at least one selected from the group consisting of a cold, asthma, sinusitis, pneumonia, sepsis, osteomyelitis, meningitis, herpes simplex, shingles, stomatitis, and psoriasis.

7. A pharmaceutical composition for preventing or treating a disease caused by immunodeficiency, characterized in that in clause 5, in addition to the extract, it further comprises a pharmaceutically acceptable carrier, excipient or diluent.

8. A pharmaceutical composition for preventing or treating a disease caused by decreased immunity, characterized in that the composition in paragraph 5 is manufactured in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.

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