Method for activating dendritic cells

Abstract

To provide a method for activating dendritic cells that can be easily ingested by a healthy human in daily life.SOLUTION: The present disclosure includes a composition for activating plasmacytoid dendritic cells (pDCs) containing young barley leaves. Preferably, dietary fibers derived from young barley leaves are used as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for activating dendritic cells, characterized by containing young barley leaves. [Background technology]

[0002] Dendritic cells (DC or DCs) are antigen-presenting cells morphologically characterized by having dendritic protrusions, and are known to play important and diverse roles. Dendritic cells include conventional dendritic cells (cDC or cDCs) and plasmacytoid dendritic cells (pDC or pDCs). cDCs directly or indirectly activate cells such as natural killer cells (NK cells), helper T cells, killer T cells, macrophages, and B cells to eliminate viruses and bacteria. It is also known that killer T cells and NK cells selectively eliminate cancer cells (Patent Document 1, Non-Patent Document 1), that interferon-γ secreted by activated NK cells has a healing effect on chronic wounds (Patent Document 2), and that macrophages remove melanin and senescent cells that have fallen into the dermis layer to achieve whitening and anti-aging effects on skin tissue (Patent Document 3, Patent Document 4). Therefore, activating cDCs exerts effects such as maintaining immune function, antiviral action, anticancer action, promoting the healing of chronic wounds, whitening action, and anti-aging action on the skin. Furthermore, since dendritic cells are essential for embryo implantation and their loss is known to cause infertility (Non-Patent Document 2), it is expected that activating cDCs may be useful in infertility treatment.

[0003] On the other hand, pDCs exhibit high production of interferon-α, a cytokine important in defense against viral infections, and have the ability to activate cells such as NK cells, helper T cells, killer T cells, macrophages, and B cells. Therefore, like cDCs, activating pDCs exerts effects such as maintaining immune function, antiviral effects, anticancer effects, promoting the healing of chronic wounds, whitening effects, and anti-aging effects on the skin.

[0004] Thus, if dendritic cells can be activated, various effects can be achieved, and therefore there has been a demand for the development of a method for activating dendritic cells. In recent years, a "dendritic cell vaccine therapy" has been developed in which dendritic cells are cultured outside the body, multiplied, and then returned to the patient's body (Non-Patent Document 3). However, since the "dendritic cell vaccine therapy" is intended to treat cancer, it cannot be performed by healthy people for the purposes of maintaining immune function in their daily lives, whitening the skin, or preventing skin aging. For this reason, there has been a demand for the development of a composition for activating dendritic cells that healthy people can easily ingest in their daily lives. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5742050 [Patent Document 2] Patent No. 4616291 [Patent Document 3] JP 2005-281205 A [Patent Document 4] Patent Publication No. 2021-195305 [Non-patent literature]

[0006] [Non-Patent Document 1] Medical Corporation Shinjukai Shin-Yokohama Kato Clinic website, "What is the mechanism of activated lymphocytes (killer T cells)?", URL: https: / / katoclinic.info / clinic-treatment / lak / system / , retrieved October 6, 2023 [Non-Patent Document 2] The Journal of Clinical Investigation, 2008, Dec; 118(12): 3954-65 [Non-Patent Document 3] Kyushu Koseikai Clinic website, Dendritic cell vaccine therapy, URL: https: / / kyushukouseikai.or.jp / dc / , retrieved October 6, 2023 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the above problems, and an object of the present invention is to provide a composition using young barley leaves and having a new function. [Means for solving the problem]

[0008] In the course of developing a material that has the effect of activating dendritic cells, the present inventors discovered that young barley leaves have an excellent effect of activating dendritic cells, which led to the completion of the present invention.

[0009] That is, the present invention is as follows. [1] A composition for activating dendritic cells, comprising young barley leaves. [2] A composition for activating dendritic cells, characterized in that the active ingredient is dietary fiber derived from young barley leaves. [3] A composition for dendritic cell activation described in either [1] or [2], characterized in that it is used for maintaining immune function. [4] A composition for dendritic cell activation described in either [1] or [2], which is a functional food. [5] The composition for activating dendritic cells according to either [1] or [2], characterized in that the dendritic cells are cDCs (conventional dendritic cells). [6] The composition for dendritic cell activation described in either [1] or [2], wherein the young barley leaves are barley leaf powder. [7] A functional food containing barley leaf powder, A functional food that claims that dietary fiber derived from young barley leaves acts on cDCs (conventional dendritic cells) and helps maintain the immune function of healthy people. [8] A functional food containing barley leaf powder, A functional food that claims that dietary fiber derived from young barley leaves acts on cDC (conventional dendritic cells). [9] A functional food as described in [8], characterized in that it is used to help maintain the immune function of healthy people.

[10] The composition for dendritic cell activation described in [1], characterized in that the daily intake amount of young barley leaves is 0.25 g or more.

[11] The composition for dendritic cell activation described in [2], characterized in that the daily intake of dietary fiber derived from young barley leaves is 0.125 g or more.

[12] Use of young barley leaves for the manufacture of a composition for activating dendritic cells.

[13] A method for activating dendritic cells in the body of an individual, comprising a step of orally ingesting young barley leaves by the individual.

[14] A composition for activating natural killer cells, helper T cells, killer T cells, B cells, or macrophages, characterized in that young barley leaves are used as an active ingredient.

[15] Use of young barley leaves for the production of a composition for activating natural killer cells, a composition for activating helper T cells, a composition for activating killer T cells, a composition for activating B cells, or a composition for activating macrophages.

[16] A method for activating natural killer cells, helper T cells, killer T cells, B cells or macrophages in the body of an individual, comprising a step of orally ingesting young barley leaves by the individual.

[17] A composition for promoting the secretion of interleukin-12 or interferon-γ, which comprises young barley leaves as an active ingredient.

[18] Use of young barley leaves for the production of a composition for promoting the secretion of interleukin-12 or a composition for promoting the secretion of interferon-γ.

[19] A method for promoting the secretion of interleukin-12 or interferon-γ in the body of an individual, comprising the step of orally ingesting young barley leaves by the individual. Effect of the Invention

[0010] The composition of the present invention can activate dendritic cells, thereby promoting the secretion of cytokines such as interleukin-12 and interferon-γ, and activating natural killer cells, helper T cells, killer T cells, B cells, and macrophages, thereby maintaining immune function and exerting effects such as antiviral and anticancer effects, promoting the healing of chronic wounds, whitening effects, and anti-aging effects on the skin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. Young barley leaves The present invention is characterized by containing young barley leaves. Barley (Hordeum vulgare L.) is an annual or biennial herb belonging to the family Poaceae, said to be native to Central Asia, and is roughly classified into two-row barley, six-row barley, etc., depending on the ear shape. Barley is often used as a grain in the form of fruit (seed), but in the present invention, young leaves are used. Comparing the two, barley fruit is mainly composed of starch (sugar), while barley leaves are mainly composed of dietary fiber and protein, and have completely different component compositions. The barley variety used for the young barley leaves of the present invention is not particularly limited as long as it is normally available, and any variety of young barley leaves, such as two-row barley or six-row barley, may be used. Note that young barley leaves refer to barley leaves before the ears begin to emerge, and in the present invention, they may include stems together with the young leaves.

[0012] As the young barley leaves, for example, crushed products, squeezed juice, extracts, extraction residues, etc. can be used. As the crushed products, dried powder (young barley leaves powder), shredded products and their dried products (dried shredded products) can be mentioned. As the squeezed juice or extracts, liquid forms are acceptable, but squeezed juice powders are preferred. As the squeezed juice powder of young barley leaves, for example, a product obtained by concentrating the squeezed juice of young leaves at low temperatures to concentrate the solid content, and freeze-drying or spray-drying the concentrate can be used. As the extract, for example, water (warm water, hot water), ethanol, and aqueous ethanol can be used. As the extraction residue, for example, a precipitate obtained by suspending young barley leaves powder in water, stirring and shaking, and then centrifuging can be used. Although the extraction residue is a fraction that is removed in the process of obtaining the squeezed juice or extract, it is rich in dietary fiber derived from young barley leaves, and therefore the effects of the present invention can be enjoyed. As the young barley leaves used in the present invention, a pulverized product is preferred, since the effects of the present invention can be more fully enjoyed by containing dietary fiber derived from young barley leaves, and the product can be produced inexpensively. Among pulverized products, a dried powder (young barley leaf powder) is particularly preferred.

[0013] In addition, barley leaf fermentation products obtained by fermenting barley leaves with microorganisms such as lactic acid bacteria are not included in the barley leaves of the present invention. When fermented, proteins, sugars, etc. are decomposed by microorganisms, and the components are chemically changed. The barley leaves after fermentation have changed into something completely different in component composition from before fermentation, and are not equivalent to the barley leaves before fermentation, so they do not fall under the barley leaves of the present invention.

[0014] When using powdered young barley leaves, for example, barley leaves that have been dried so that the moisture content of the young barley leaves is 10% by mass or less, preferably 5% by mass or less, can be used.

[0015] The young barley leaf powder used in the present invention preferably has a median diameter of 5 μm to 50 μm, more preferably 8 μm to 30 μm. The median diameter means the particle diameter at which the cumulative 50% of the particle size distribution of the powder is reached, and specifically, it is the particle diameter at which the cumulative 50% (×50) of the particle size distribution is reached, as measured using a laser diffraction / scattering light type particle size distribution measuring device, LMS-300 or LMS-350, manufactured by Seishin Enterprise Co., Ltd.

[0016] Moreover, the barley leaves used in the present invention preferably contain 10% by mass or more of dietary fiber, more preferably 20% by mass or more, and even more preferably 30% by mass or more, so that the effects of the present invention can be more effectively enjoyed. The upper limit of the dietary fiber content may be, for example, 80% by mass. The dietary fiber referred to here is the total amount of insoluble dietary fiber and soluble dietary fiber. The amount of dietary fiber is measured by the Prosky method.

[0017] Furthermore, in order to obtain the effects of the present invention more effectively, the young barley leaves used in the present invention preferably contain 5% by mass or more of insoluble dietary fiber, more preferably 10% by mass or more, and even more preferably 20% by mass or more. The upper limit may be, for example, 70% by mass. The amount of insoluble dietary fiber is measured by the modified Prosky method.

[0018] In order to set the dietary fiber content and insoluble dietary fiber content within the above ranges, the barley leaf powder may be prepared by crushing the barley leaves without squeezing or extracting them in the production method of the barley leaves.

[0019] 2. Dendritic Cells In the present invention, dendritic cells are a concept that encompasses conventional dendritic cells (cDC or cDCs) and plasmacytoid dendritic cells (pDC or pDCs). In the present invention, activation of dendritic cells means that some function of dendritic cells is enhanced, or the amount of genes, proteins, etc. expressed in dendritic cells is amplified. Specific examples include promotion of dendritic cell maturation, enhancement of antigen uptake ability, enhancement of antigen presentation ability, T cell activation ability, and enhancement of cytokine production such as interleukin 12. The composition for dendritic cell activation of the present invention is particularly excellent in activating cDCs (conventional dendritic cells).

[0020] When cDCs are activated, the expression of MHC (short for Major Histocompatibility Complex; in humans, it is called HLA (Human Leukocyte Antigen)) is enhanced, and the production of interleukin-12 (IL-12) is also enhanced. IL-12 is a protein with a molecular weight of approximately 70,000, and is responsible for inducing differentiation of naive T cells into helper T cells (T cell activation) and activating NK cells. Helper T cells activated by IL-12 produce interferon gamma (IFN-γ). IFN-γ is a dimeric protein that conveys instructions from helper T cells to killer T cells and B cells. By receiving this instruction, killer T cells and B cells are activated and can attack pathogens such as viruses and bacteria. In addition, IFN-γ activates macrophages, which promote the macrophages to eat bacteria that have invaded the body, melanin that has fallen into the dermis layer, senescent cells, etc., and digest and kill them. In this way, when cDCs are activated, the secretion of interleukin-12 is promoted, activating helper T cells, and the secretion of interferon-γ from helper T cells is promoted, activating natural killer cells, killer T cells, B cells, and macrophages. On the other hand, pDCs produce interferon alpha (IFN-α), a cytokine that is important in defense against viral infections. IFN-α is a signaling substance that activates various immune cells, and the production of IFN-α activates natural killer cells, helper T cells, killer T cells, B cells, and macrophages. By activating dendritic cells through the above-mentioned mechanism, natural killer cells, helper T cells, killer T cells, B cells and macrophages are activated, resulting in effects such as maintaining immune function, antiviral effects, anticancer effects, promoting the healing of chronic wounds, whitening effects and anti-aging effects on the skin.

[0021] 3. Composition of the Present Invention <Composition for activating dendritic cells>

[0022] The composition for dendritic cell activation of the present invention is not particularly limited as long as it contains young barley leaves and can be distinguished from other products as a product in that it is used for activating dendritic cells. For example, the scope of the present invention includes a product that displays the function of dendritic cell activation on the main body, packaging, instructions, or promotional materials (advertising media) of the product according to the present invention. The composition for dendritic cell activation of the present invention may display young barley leaves or a component derived from young barley leaves (such as dietary fiber derived from young barley leaves) as an active ingredient, but is not limited to a product that displays young barley leaves or a component derived from young barley leaves (such as dietary fiber derived from young barley leaves) as an active ingredient on the packaging of the product. For example, the active ingredient may not be specified. Note that a composition for dendritic cell activation containing dietary fiber derived from young barley leaves as an active ingredient may contain dietary fiber derived from young barley leaves, and fractionated dietary fiber derived from young barley leaves may not be used. For example, by blending barley leaves containing dietary fiber derived from barley leaves, such as barley leaves powder (ground powder), a composition for dendritic cell activation containing dietary fiber derived from barley leaves as an active ingredient can be obtained. In addition, even if it is a general food, it is within the scope of the present invention if it is manufactured and sold with a suggested use. For example, foods sold with a personal experience of ingesting the food, which is described on a website, etc., as a personal impression of the person who ingested the food, and which mentions the activation of dendritic cells, are also within the scope of the present invention. In addition, functional foods that use papers showing the activation of dendritic cells by barley leaves as the scientific basis of functionality, have barley leaves or ingredients derived from barley leaves as functional ingredients, and report the functionality related to the maintenance of immune function are also within the scope of the present invention. In this application, the maintenance of immunity is a concept that includes suppression of the decline of immune function and / or improvement (improvement) of immune function.

[0023] Specifically, in the case of so-called health foods, the following claims are made: "activates dendritic cells," "activates cDCs," "activates pDCs," "helps to activate dendritic cells," "helps to activate cDCs," "helps to activate pDCs," "helps to activate dendritic cells," "helps to activate cDCs," "helps to activate pDCs," "activates dendritic cells," "activates cDCs," "activates pDCs," "helps to activate dendritic cells," "helps to activate cDCs," "helps to activate pDCs," "helps to activate dendritic cells," "helps to activate cDCs," "helps to activate pDCs," "acts on dendritic cells," "acts on cDCs," "acts on pDCs," "helps the function of dendritic cells," "helps the function of cDCs," "helps the function of pDCs," "acts on dendritic cells and helps to maintain the immune function of healthy people," "acts on cDCs (conventional dendritic cells) and helps to maintain the immune function of healthy people," "acts on pDCs (plasmacytoid dendritic cells) and helps to improve the immune function of healthy people," "It helps maintain the immune function of healthy people by supporting the function of dendritic cells," "It helps maintain the immune function of healthy people by acting on cDC (conventional dendritic cells)," "It helps maintain the immune function of healthy people by acting on pDC (plasmacytoid dendritic cells)," "It helps maintain the immune function of healthy people by influencing the function of dendritic cells," "It helps the function of cDC (conventional dendritic cells) and helps maintain the immune function of healthy people," "It helps the function of pDC (plasmacytoid dendritic cells) and helps maintain the immune function of healthy people," "It acts on dendritic cells "Affects cDC (conventional dendritic cells) and helps maintain immune function in healthy people", "Affects pDC (plasmacytoid dendritic cells) and helps maintain immune function in healthy people", "Affects dendritic cells and helps maintain immune function", "Affects cDC (conventional dendritic cells) and helps maintain immune function", "Affects pDC (plasmacytoid dendritic cells) and helps maintain immune function", "Helps dendritic cells function and helps maintain immune function", "Affects cDC (conventional dendritic cells) and helps maintain immune function",Examples of products that display the following statements include "Helps maintain immune function by acting on pDC (plasmacytoid dendritic cells)", "Helps maintain immune function by influencing the function of dendritic cells", "Helps maintain immune function by supporting the function of cDC (conventional dendritic cells)", "Helps maintain immune function by supporting the function of pDC (plasmacytoid dendritic cells)", "Helps maintain immune function by acting on dendritic cells", "Helps maintain immune function by acting on cDC (conventional dendritic cells)", "Helps maintain immune function by acting on pDC (plasmacytoid dendritic cells)", etc.

[0024] As described above, the composition for dendritic cell activation of the present invention exhibits effects such as maintaining immune function, antiviral effect, anticancer effect, promoting the healing of chronic wounds, whitening effect, and anti-aging effect of the skin by promoting the secretion of interleukin-12 and the like and activating natural killer cells, etc. Therefore, the composition for dendritic cell activation of the present invention can be used for the purposes of maintaining immune function, antiviral effect, anticancer effect, promoting the healing of chronic wounds, whitening effect, and anti-aging effect of the skin.

[0025] According to the composition of the present invention, dendritic cells are activated, thereby promoting the secretion of interleukin-12 and the secretion of interferon-γ from activated helper T cells. Therefore, the composition of the present invention can be used as a composition for promoting the secretion of interleukin-12 or interferon-γ. Furthermore, according to the composition of the present invention, dendritic cells are activated, thereby activating natural killer cells, helper T cells, killer T cells, B cells, and macrophages. Therefore, the composition of the present invention can be used as a composition for activating natural killer cells, a composition for activating helper T cells, a composition for activating killer T cells, a composition for activating B cells, or a composition for activating macrophages.

[0026] <Formulation of the composition, etc.> The composition of the present invention can be used as an oral composition. The form of the oral composition is not particularly limited, and examples thereof include food and drink, quasi-drugs, and medicines, but it is preferable to use it as a food and drink from the viewpoint of daily intake of barley leaves to supplement daily nutrition. The form of the food and drink composition is not particularly limited, and examples thereof include functional food, food for specified health use, health food, and general food and drink, but from the viewpoint of making it easy for consumers to understand the effects of the present invention, functional food and food for specified health use are preferred, and functional food is most preferred.

[0027] The form of the composition of the present invention may be, for example, powder, granule, liquid, tablet, capsule, etc. It may also be in the form of jelly, cookies, etc. As described above, the young barley leaves of the present invention are not fermented and have a good taste, so they are suitable for ingestion in a form that allows the taste to be felt. Therefore, the forms of powder, granule, liquid, and tablet are preferred, and the forms of powder, granule, and liquid are particularly preferred.

[0028] The content of young barley leaves in the composition of the present invention may be appropriately determined within a range that exhibits the desired effect. For example, the composition of the present invention may contain young barley leaves in an amount of 0.01 to 100% by mass, preferably 0.1 to 100% by mass, and more preferably 1 to 100% by mass, calculated on a dry mass basis.

[0029] The content of dietary fiber derived from young barley leaves in the composition of the present invention may be appropriately determined within a range that exhibits its effect. For example, the dietary fiber derived from young barley leaves may be contained in the composition of the present invention in an amount of 0.01 to 80% by mass, preferably 0.1 to 75% by mass, and more preferably 1 to 70% by mass, calculated on a dry mass basis.

[0030] In the present invention, the intake amount of young barley leaves is not particularly limited, but in order to enjoy the effects of the present invention more effectively, the intake amount of young barley leaves is preferably 0.25 g or more, more preferably 0.5 g or more, and particularly preferably 1.0 g or more per day for an adult. The upper limit is, for example, 30 g, preferably 20 g, and more preferably 10 g.

[0031] In addition, the intake amount of dietary fiber derived from young barley leaves in the present invention is not particularly limited, but in order to enjoy the effects of the present invention more effectively, the intake amount of dietary fiber derived from young barley leaves is preferably 0.125 g or more, more preferably 0.25 g or more, and particularly preferably 0.5 g or more per day for an adult. The upper limit is, for example, 15 g, preferably 10 g, and more preferably 5 g.

[0032] The composition of the present invention may be appropriately designed so that the daily intake of young barley leaves or dietary fiber derived from young barley leaves is the above-mentioned intake amount, and may be in a form in which the above-mentioned intake amount is taken in one sitting, or in a form in which the above-mentioned intake amount is taken in multiple sittings. That is, for example, the daily amount can be stored in one container, or divided into 2 to 4 multiple containers.

[0033] The composition of the present invention can be produced by a known method, adding ingredients other than young barley leaves as necessary. EXAMPLES

[0034] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be achieved.

[0035] Test 1. Evaluation of dendritic cell activation by young barley leaves To evaluate the dendritic cell activation effect of young barley leaves, the gene amounts of cytokines secreted from dendritic cells were evaluated using dendritic cells differentiated from mouse bone marrow cells in the test described below.

[0036] Test 1(A). Evaluation of TNF-α gene expression Tumor necrosis factor-α (TNF-α) was used as a cytokine indicator. TNF-α is one of the powerful mediators of biological responses that is rapidly secreted from cDCs and pDCs upon invasion by foreign bodies, and serves as an indicator of dendritic cell activation.

[0037] <Test substance> (Young barley leaf powder) The raw material used was the above-ground part (leaves and stems) of barley harvested at a height of about 30 cm. The barley was washed with water to remove any adhering mud, cut into pieces of about 5 cm in size, and then blanched once in 95°C hot water for about 100 seconds, and cooled in cold water. The resulting young barley leaves were dried with hot air in a dryer and coarsely crushed to a size of about 1 mm. The barley leaves were then finely crushed using a jet mill grinder to produce young barley leaf powder (median diameter about 20 μm, moisture content 5% by mass or less, dietary fiber derived from young barley leaves 40% by mass).

[0038] (Test substances other than barley leaf powder) The following test substances were used other than barley leaf powder: Young barley leaf juice powder: A powder made by mixing young barley leaf juice with dextrin and drying the mixture (2% by mass of dietary fiber derived from young barley leaves) was used. Cellulose: Commercially available cellulose was used. Vitamin D3: Vitamin D3 commercially available as a reagent was used. β-glucan (derived from barley seeds): A commercially available β-glucan (purified product derived from barley seeds; purity 98%) was used as a reagent.

[0039] <Test Method> After dissection of C57BL / 6J mice (8 weeks old), bone marrow mononuclear cells were collected from the femur and tibia and cultured using differentiation induction medium (RPMI1640 medium containing 10% fetal bovine serum, 100 μM 2-mercaptoethanol, and 100 ng / mL Flt3 ligand). Specifically, the cells were seeded at 100 μL / well in a 96-well plate to give 3×105 cells / well, and differentiated into bone marrow-derived dendritic cells (including both cDCs and pDCs) by culturing for 7 days in a 5% CO2 incubator (Astec CO2 incubator) at 37°C while changing the medium every 3 to 4 days. After removing the medium from each well, 100 μL / well of differentiation induction medium containing 0.5% DMSO was added to each well so that the total concentration of the test substance was 10 μg / mL (for barley leaf juice powder, the concentration of barley leaf juice components excluding dextrin was 10 μg / mL), and the cells were cultured for 72 hours. The proportions of each test substance are shown in Table 1. Then, the medium was removed from each well, and RNA was extracted using an RNeasy Mini Kit (QIAGEN). The RNA was then subjected to PCR using the One Step TB Green PrimeScript RT-PCR Kit II.<Perfect Real Time> The gene expression level (mRNA expression level) of TNF-α was measured by real-time PCR using the primers Mm_Il12B_1_SG QuantiTect Primer Assay and Mm_Tnf 1_SG QuantiTect Primer Assay (QIAGEN).

[0040] <Test Results> The TNF-α gene expression levels (relative values) of the Examples and Comparative Examples were evaluated with the value (TNF-α / β-actin) of the control where no test substance was added being set at 1. The results are shown in Table 1. In Examples 1 and 2, which contained young barley leaves, the TNF-α gene expression level was increased compared to the control. Since TNF-α is an indicator of dendritic cell activation, it was suggested that young barley leaves activate dendritic cells. In particular, in Example 1, which contains young barley leaf powder, the promotion of TNF-α gene expression was more pronounced. Dietary fiber (cellulose) and vitamin D, which are known to have immunostimulatory effects, did not promote TNF-α gene expression (Comparative Examples 1 and 2). This confirmed that ingredients with immunostimulatory effects do not necessarily activate dendritic cells.

[0041] [Table 1]

[0042] Study 1(B). Evaluation of IL-12 gene expression As a cytokine, IL-12 (interleukin 12) was used as an indicator. As described above, IL-12 is a cytokine secreted by cDC (conventional dendritic cells) and serves as an indicator of dendritic cell activation.

[0043] <Test substance> The same young barley leaf powder as in Test 1(A) was used.

[0044] <Test Method> Bone marrow mononuclear cells were collected from the femur and tibia of 8-week-old C57BL / 6J mice and cultured in differentiation induction medium (RPMI1640 medium containing 10% fetal bovine serum, 100 μM 2-mercaptoethanol, 100 ng / mL Flt3 ligand, 100 units / mL penicillin, and 100 μg / mL streptomycin) to induce bone marrow-derived dendritic cells. After 7 days of culture, a differentiation induction medium containing barley leaf powder at a final concentration of 10 μg / mL or a differentiation induction medium without barley leaf powder as a control was added. After 72 hours of culture, RNA was collected using RNeasy Mini Kit (QIAGEN) and real-time PCR was performed using One Step TB Green (registered trademark) PrimeScript RT-PCR Kit II (Perfect Real Time) (TaKaRa) to evaluate the mRNA expression level of IL-12. The mRNA expression level of β-actin was used as an endogenous control. The primers used were Mm_Il12B_1_SG QuantiTect Primer Assay and Mm_Actb_1_SG QuantiTect Primer Assay (QIAGEN).

[0045] <Test Results> The IL-12 gene expression level (relative value) was evaluated with the control value (IL-12 / β-actin) of no test substance added set as 1. The results are shown in Table 2. Compared to the control, the IL-12 gene expression level was increased in the group to which young barley leaf powder was added, confirming that dendritic cells had been activated.

[0046] [Table 2]

[0047] Test 2. Evaluation of dendritic cell activation by dietary fiber derived from young barley leaves The results shown in Test 1(A) showed that barley leaf powder (ground barley leaf powder) had a higher dendritic cell activation effect than barley leaf juice powder, which is a concentrated form of the water-soluble components of barley leaves. From these results, it was inferred that barley leaf-derived dietary fiber, which is removed during the process of squeezing the barley leaves, may contribute to dendritic cell activation. To confirm this, the dendritic cell activation effect of barley leaf-derived dietary fiber was evaluated by the following test.

[0048] Test 2 (A) Evaluation of IL-12 gene expression in dendritic cells To evaluate the dendritic cell activation effect of dietary fiber derived from young barley leaves, the gene amounts of cytokines secreted from dendritic cells were evaluated using the test described below. IL-12 (interleukin 12) was used as an indicator of cytokines.

[0049] <Test substance> Dietary fiber derived from young barley leaves was fractionated from the young barley leaves powder obtained by the method described in Test 1 and used as the test substance.

[0050] <Test Method> Bone marrow mononuclear cells were collected from the femur and tibia of C57BL / 6J mice (7 weeks old) and cultured in differentiation induction medium (RPMI1640 medium containing 10% fetal bovine serum, 55 μM 2-mercaptoethanol, 20 ng / mL GM-CSF, 1% penicillin, and 10 mM HEPES) to induce bone marrow-derived dendritic cells. After culturing for 6 days in a 5% CO2 incubator at 37°C, test substance-added medium containing dietary fiber derived from barley young leaves (RPMI1640 medium containing 10% fetal bovine serum, 55 μM 2-mercaptoethanol, 1% penicillin, and 10 mM HEPES), test substance-added medium not containing dietary fiber derived from barley young leaves (control), and test substance-added medium containing zymosan (positive control) were prepared. The concentrations of each test substance are shown in Table 3. After 5 hours of culture, RNA was collected using RNeasy Mini Kit (QIAGEN), and real-time PCR was performed using One Step TB Green (registered trademark) PrimeScript (trademark) RT-PCR Kit II (Perfect Real Time) (manufactured by TaKaRa) to evaluate the gene expression level (mRNA expression level) of IL-12. The mRNA expression level of Rn18s was used as an endogenous control. The primers used were Mm_Il12B_1_SG QuantiTect Primer Assay and Mm_Rn18S_3_SG QuantiTect Primer Assay (manufactured by QIAGEN).

[0051] <Test Results> The IL-12 gene expression levels (relative values) of the Examples and Controls were evaluated with the value (IL-12 / Rn18s) of the control without addition of the test substance set at 1. The results are shown in Table 3. The IL-12 gene expression level increased with dietary fiber derived from young barley leaves, and the expression level was significantly higher than that with zymosan, a known dendritic cell activator. This result suggests that dietary fiber derived from young barley leaves contributes to dendritic cell activation as an active ingredient of young barley leaves.

[0052] [Table 3]

[0053] Test 2 (B) Evaluation of IL-12 production in dendritic cells In order to confirm the correlation between the gene and protein expression levels of IL-12, the amount of IL-12 (amount as protein) secreted from dendritic cells was evaluated in the test described below using the dietary fiber derived from young barley leaves prepared in Test 2(A).

[0054] <Test Method> Dendritic cells were differentiated using bone marrow mononuclear cells collected from C57BL / 6J mice (8 weeks old) in the same manner as in Test 2(A). After 6 days of culture in a 5% CO2 incubator at 37°C, test substance-added media containing dietary fiber derived from young barley leaves and test substance-added media not containing dietary fiber derived from young barley leaves (control) were prepared to the final concentrations shown in Table 4. After 48 hours of culture, the culture supernatant was collected and the amount of IL-12 was evaluated using the Mouse IL-12p40 SimpleStep ELISA Kit (Abcam).

[0055] <Test Results> The measured IL-12 concentrations are shown in Table 4. Comparing IL-12 concentrations, the control was 17.2 pg / mL, whereas Examples 6-8 were 75.7 pg / mL (4.4 times the control), 140.7 pg / mL (8.2 times the control), and 255.2 pg / mL (14.8 times the control), respectively. These results suggest that there is a correlation between the expression levels of the IL-12 gene and protein, and that dietary fiber derived from young barley leaves activates dendritic cells, resulting in increased IL-12 production.

[0056] [Table 4]

[0057] Test 3. Evaluation of natural killer cell activation by barley leaves Young barley leaves are thought to activate natural killer cells (NK cells) by activating dendritic cells and promoting the secretion of IL-12. However, it was unclear whether young barley leaves also act directly on NK cells. To confirm this, the cytotoxic activity of NK cells (the ability of NK cells to kill target cells) was evaluated using the test described below.

[0058] <Test substance> For the spirulina, crushed powder of spirulina was used. Other test substances were the same as those used in Test 1(A).

[0059] <Test Method> After dissection of C57BL / 6J mice (8 weeks old), spleen cells were isolated, and the above cells were diluted with 10% fetal bovine serum-RPMI1640 medium to 5×106 cells / well, and the cell suspension was seeded at 100 μL / well in a 96-well plate. 100 μL / well of medium (0.5% DMSO-10% fetal bovine serum-RPMI1640 medium) was added to the seeded cells so that the total concentration of the test substance was 1 μg / mL (for barley leaf juice powder, the concentration of barley leaf juice components excluding dextrin was 1 μg / mL), and the cells were cultured for 18 hours in a 5% CO2 incubator at 37°C (Astec CO2 incubator). The proportions of each test substance are shown in Table 5. After the incubation, the plate was centrifuged (1000 rpm, 10 min) using a centrifuge. 150 μL of the supernatant was removed, and 150 μL of 1% BSA-RPMI1640 was added. This was repeated again. Then, NK cell-sensitive YAC-1 cells (calcein stained) were added as target cells and incubated for 4 hours. After the culture, the plate was centrifuged (1000 rpm, 10 min) using a centrifuge, and the supernatant was collected in a 96-well black plate. The fluorescence intensity (excitation wavelength 490 nm, fluorescence wavelength 515 nm) of the collected supernatant was measured using a microplate reader (Molecular Devices' "SpectraMax i3x") to evaluate the NK cytotoxicity.

[0060] <Test Results> Table 5 shows the NK cytotoxic activity (relative value) with the control value taken as 1. The higher the relative value of the NK cytotoxic activity, the more activated the NK cells are. In Examples 9 and 10, which contain young barley leaves, the NK cytotoxic activity was higher than that of the control. This suggests that young barley leaves not only activate NK cells by promoting the secretion of IL-12 through dendritic cell activation, but also act directly on NK cells to activate them. Note that the addition of dietary fiber (cellulose), vitamin D, or spirulina, which are known to have immunostimulatory effects, did not activate NK cells.

[0061] [Table 5]

[0062] Test 4. Evaluation of dendritic cell activity and NK cell activity through clinical trials In order to confirm whether dendritic cells and NK cells are activated when humans ingest young barley leaves, the clinical trial described below was conducted.

[0063] <Test Method> 1) Test food The test food was a powdered drink made by mixing young barley leaf powder (manufactured using the method described in Study 1) with maltose. The control food was a powdered drink that did not contain young barley leaf powder, but was made by mixing maltose with coloring and flavoring to make it indistinguishable in appearance from the test food. The daily intake of both the test food and control food was designed to be 5.4g, and 2.7g was distributed to the subjects in plain aluminum bags to ensure blinding of the subjects and the interventionists. The calorie and nutritional values ​​of the test food per daily intake (2 bags) are shown in Table 6. The test food contained 0.7g of dietary fiber derived from young barley leaves per daily intake (2 bags).

[0064] [Table 6]

[0065] 2) Subjects Paid volunteers were recruited from the public, and 56 people (19 men and 37 women) who met the following inclusion criteria and did not violate the exclusion criteria were enrolled as subjects by the principal investigator. In addition, the subjects were fully explained about the study before the start of the study, and written consent was obtained. Inclusion criteria: (1) Healthy adult men and women aged 20 years or older and under 65 years old at the time of obtaining consent, (2) Those who have been fully informed of the purpose and content of the study, have the capacity to consent, fully understand the study, voluntarily volunteer to participate, and agree to participate in the study in writing. Exclusion criteria: (1) Individuals suffering from, undergoing treatment for, or with a history of serious diseases such as diabetes, kidney disease, liver disease, or heart disease, thyroid disease, adrenal gland disease, or other metabolic diseases; (2) Individuals with a chronic disease and regular use of medication; (3) Individuals with a digestive disease or history of digestive surgery that may affect digestion and absorption; (4) Individuals who are deemed unsuitable as study subjects based on the blood test administered for screening; (5) Individuals who have never experienced symptoms of upper respiratory tract infection in the past two winters; (6) Individuals suffering from hay fever or allergic rhinitis and taking medication; (7) Individuals who are unable to stop taking supplements or health foods (including functional foods) that may affect the immune system; (8) Individuals who consume approximately 6000 mg of pure alcohol per day. (9) smokers; (10) those who engage in strenuous exercise such as running or soccer; (11) those who cannot abstain from alcohol from the day before the screening test or each test; (12) those who have declared allergies to the ingredients of the study food; (13) those with a history or current medical history of drug or alcohol dependence; (14) shift or night shift workers; (15) those planning to travel abroad, including overseas trips; (16) pregnant or breastfeeding women, or those who intend to become pregnant; (17) those who have used drugs that are thought to affect immune function (including antiallergy drugs and antibiotics) within 2 weeks of the screening test; (18) those who have donated blood or blood components exceeding 200 mL within 4 weeks of the screening test, or 400 mL within 3 months; (19) those who have participated in another study within 1 month of obtaining consent, or those who intend to participate; (20) those who are otherwise deemed unsuitable as study subjects by the principal investigator

[0066] 3) Test method This study was conducted as a placebo-controlled, randomized, double-blind, parallel-group comparative study (allocation ratio: 1:1) for a total of 9 weeks, consisting of a pre-observation period (1 week) and an intake period (8 weeks). The principal investigator enrolled the subjects according to the inclusion and exclusion criteria, and the statistical analysis manager allocated them using a block randomization method (block size 4) with gender, age, and cDC activity markers as adjustment factors. A controller who was not directly involved in the study assigned the two groups to the test food group and the control food group. In addition, the controller created and sealed a table (key code) listing the allocation results, and kept it sealed until the key code was disclosed after the analysis subjects were determined, ensuring blinding. During the study period, the subjects were instructed to consume two bags (5.4 g) of the test food (test food for the test food group and control food for the control food group) per day dissolved in an appropriate amount of water or hot water. During the study period, subjects were instructed to live the same lifestyle as before the study began, to avoid using supplements and health foods, to not change the frequency of intake for those who regularly consume foods containing lactobacillus or bifidobacteria, to avoid excessive alcohol intake, and to avoid participating in other studies. In addition, subjects were instructed to avoid alcohol intake from the day before all tests, to get enough sleep the day before all tests, to avoid eating and drinking anything other than water after 9 p.m. the day before all tests, and to avoid strenuous exercise from the day before all tests until the end of the tests. Subjects were to use medicines with the permission of the principal or co-investigator, except in emergency cases.

[0067] 4) Inspection items Measurements of cDC activity and activated NK cells were conducted twice: once before intake (hereafter, "before intake") and once eight weeks after intake (hereafter, "after eight weeks after intake"). cDC activity was evaluated by measuring HLA-DR, a cDC activity marker, in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood and stained with CD4, CD141, CD11c, and HLA-DR antibodies, and measured using a flow cytometer. Among CD4-positive cells, cells positive for both CD141 and CD11c were considered cDCs, and the mean fluorescence intensity (MFI) of HLA-DR was calculated. Activated NK cells were evaluated by measuring CD69-positive cells, an activation marker for NK cells. PBMCs were isolated from blood, stained with CD3, CD45, CD56, and CD69 antibodies, and measured using a flow cytometer. CD3-negative cells and CD45, CD56, and CD69-positive cells were defined as activated NK cells, and the percentage of activated NK cells in lymphocytes was calculated.

[0068] 5) Subjects of analysis The study enrolled 56 subjects (19 men and 37 women), with no dropouts after randomization and 56 subjects starting the study. During the study period, 4 subjects dropped out because the principal investigator determined they met the discontinuation criteria, leaving 52 subjects who completed the study. The reasons and numbers that met the discontinuation criteria are as follows: 1 subject (test food group) became ill for reasons other than the test food and found it difficult to continue the study, 1 subject (control food group) was judged to have engaged in behavior that undermined his reliability as a subject, and 2 subjects (test food group) did not come to the hospital on the day of the test. After the study was completed, eight subjects met the rejection criteria, leaving 44 subjects (15 men and 29 women) for analysis. The reasons for the rejection criteria and the number of subjects were as follows: (1) Seven subjects were found to have violated the precautions during the study (changes in sleep habits: one in the test food group, one in the control food group; changes in daily rhythm (work environment): one in the test food group, four in the control food group), (2) One subject (one in the test food group) developed a new illness (hay fever) during the study period.

[0069] <Test Results> The results of the analysis of the expression level of HLA-DR, a cDC activity marker, and the percentage of activated NK cells are shown in Table 7. The rate of change in both HLA-DR expression level and the percentage of activated NK cells was significantly higher in the test food group than in the control food group after 8 weeks of ingestion. These results confirmed that oral ingestion of young barley leaves by humans activates dendritic cells and NK cells.

[0070] [Table 7]

[0071] Test 5. Production of powdered beverages The powdered beverage of Example 11 was manufactured from the following ingredients. Specifically, the raw materials were mixed, and then fluidized bed granulation was performed using a granulator to manufacture a granular powdered beverage. The manufactured powdered beverage was filled into an aluminum pouch so that each bag contained 3 g. The obtained powdered beverage may be suspended in about 80 to 150 mL of water before consumption. By taking one packet of the powdered beverage of Example 11 per day (0.3 g as young barley leaf powder, 0.135 g as dietary fiber derived from young barley leaves), dendritic cells are activated, and the effects of the present invention are achieved. [Example 11] Barley grass powder (contains 45% by mass of dietary fiber) 10% by mass Matcha 4% by mass Reduced maltose 86% by mass [Industrial Applicability]

[0072] The composition of the present invention contains young barley leaves and therefore exerts an excellent dendritic cell activation effect, thereby enabling the maintenance of immune function, and further exerts effects such as antiviral and anticancer effects, an effect of promoting the healing of chronic wounds, a skin whitening effect, and an anti-aging effect on the skin. Since it can be used as a food or drink intended for dendritic cell activation, it has high industrial usefulness.

Claims

1. A composition for activating plasmacytoid dendritic cells (pDC), comprising young barley leaves.

2. A composition for activating plasmacytoid dendritic cells (pDC), characterized in that dietary fiber derived from young barley leaves is an active ingredient.

3. A composition for activating natural killer cells, comprising young barley leaves.

4. A composition for activating natural killer cells, characterized in that dietary fiber derived from young barley leaves is an active ingredient.

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